Leaf architecture directly influences canopy structure, consequentially affecting yield. We discovered a maize (Zea mays) mutant with aberrant leaf architecture, which we named drooping leaf1 (drl1). Pleiotropic mutations in drl1 affect leaf length and width, leaf angle, and internode length and diameter. These phenotypes are enhanced by natural variation at the drl2 enhancer locus, including reduced expression of the drl2-Mo17 allele in the Mo17 inbred. A second drl2 allele, produced by transposon mutagenesis, interacted synergistically with drl1 mutants and reduced drl2 transcript levels. The drl genes are required for proper leaf patterning, development and cell proliferation of leaf support tissues, and for restricting auricle expansion at the midrib. The paralogous loci encode maize CRABS CLAW co-orthologs in the YABBY family of transcriptional regulators. The drl genes are coexpressed in incipient and emergent leaf primordia at the shoot apex, but not in the vegetative meristem or stem. Genome-wide association studies using maize NAM-RIL (nested association mapping-recombinant inbred line) populations indicated that the drl loci reside within quantitative trait locus regions for leaf angle, leaf width, and internode length and identified rare single nucleotide polymorphisms with large phenotypic effects for the latter two traits. This study demonstrates that drl genes control the development of key agronomic traits in maize.

email byang@iastate.edu) † These two authors contribute equally to this work.Keywords: TAL effector nuclease, gene editing, targeted mutagenesis, maize, Glossy2. SummaryTranscription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono-or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

Caridean shrimp are critical components of sea-grass communities and occasionally harbor parasitic bopyrid isopods, which can negatively impact their hosts. However, the ecological factors that drive infection rates of parasitic bopyrid isopods in host carideans are poorly known. We examined 43,875 carideans belonging to 6 families and 11 genera from 19 shallow-water localities throughout south Florida. Of these, only 114 shrimp (belonging to 5 genera) were found to be infested with bopyrids (an additional 251 had deformed carapaces consistent with recent infestation). We identified 13 bopyrid species ( Bopyrina abbreviata, Bopyrinella thorii, Eophrixus subcaudalis, Loki circumsaltanus, Metaphrixus carolii, Ovobopyrus alphezemiotes, Parabopyrella lata, Parabopyrella richardsonae, Parabopyriscus stellatus, Capitetragonia alphei, Probopyrus pandalicola, Schizobopyrina urocaridis, and an unidentified Diplophryxus sp.). Bopyrid infection rates were very low throughout the study area, with mean prevalence of 0.26% (range 0.04-1.48%). Furthermore, each isopod species was only ever recovered from a single host genus, suggesting a high degree of genus-level specificity. At the community level, multivariate analyses (RELATE and BVSTEP) indicated that bopyrid community composition was correlated with host community structure, latitude, and temperature, as well as the relative coverage of the sea grasses Thalassia sp. and Syringodium sp. and the alga Penicillus sp. Only 4 parasite taxa were sufficiently abundant to warrant further analysis at the individual taxon level: B. abbreviata, B. thorii, Diplophryxus sp., and P. pandalicola; stepwise regression indicated that bopyrid infection rates were primarily driven by the abundance of their specific hosts, and secondarily by environmental variables such as temperature and depth, as well as algal and sea-grass community composition.

Grass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize (Zea mays) RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in sorghum (Sorghum bicolor, Sb) and green millet (Setaria viridis, Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the non-frameshifted downstream copy, complemented ra1-R branching defects and induced unusual fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of an SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture.

Grass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in Sorghum bicolor (Sb) and Setaria viridis (Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the non-frameshifted downstream copy, complemented ra1-R branching defects and induced novel fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of an SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture.