Carol Auer

Crop improvement using small RNAs: applications and predictive ecological risk assessments

Crops can be modified by engineering novel RNA interference (RNAi) pathways that create small RNA molecules to alter gene expression in crops or plant pests. RNAi can generate new crop quality traits or provide protection against insects, nematodes and pathogens without introducing new proteins into food and feed products. As a result, stakeholders and regulators need to construct credible ecological risk assessments (ERAs) that characterize potential exposure pathways and hazards for RNAi crops, including off-target effects, non-target effects and impacts from genetic mutations and polymorphisms. New methods are needed to identify RNAi crops and measure the environmental persistence of small RNAs. With some modifications, it seems likely that current ERA frameworks can be applied to most crops engineered through RNAi.

Cytokinin conjugation: recent advances and patterns in plant evolution

Cytokinin (CK) conjugates are important in plant development because they regulate active CK concentrations, CK transport, storage, and irreversible inactivation. While numerous CK conjugates have been identified in higher plants, the biological functions of these compounds, their location within cells and tissues, and the enzymes and genes involved in their regulation are not clearly understood. In this paper, recent advances are reported which have occurred through the study of transgenic plants containing the ipt or rolC genes, the identification of new regulatory enzymes affecting CKs, and the characterization of new CK conjugates. In addition, a survey of the literature is presented which examines the pattern of CK conjugates found in different plant taxa. Based on current knowledge, it appears that green algae, mosses, and ferns contain relatively few CK conjugates of isopentenyl adenine (iP) and zeatin (Z). In contrast, higher land plants, such as gymnosperms and angiosperms, contain a more complex set of CKs, primarily conjugates of Z and dihydrozeatin (DHZ). This suggests that the pattern of CK conjugation has become more complex in parallel with the increasing complexity of higher plants.

Ecological Risk Assessment and Regulation for Genetically-Modified Ornamental Plants

Classic plant breeding has increased the beauty and utility of ornamental plants, but biotechnology can offer completely new traits for plants used in homes and gardens. The creation of blue petal color in carnations and roses are examples where biotechnology has created novelty that conventional hybridization cannot match. However, all innovations have benefits and risks, and future commercialization of transgenic ornamental plants raises complex questions about potential negative impacts to managed landscapes and natural ecosystems. Predictive ecological risk assessment is a process that uses current knowledge to estimate future environmental harms or benefits arising from direct or indirect exposure to a genetically-modified (GM) plant, its genes, or gene products. This article considers GM ornamental plants in the context of current ecological risk assessment principles, research results, and current regulatory frameworks. The use of ecological risk assessment by government agencies to support decision-making is reviewed in the context of ornamental plants. Government risk assessments have usually emphasized the potential for pollen-mediated gene flow, weediness in managed areas, invasion of natural areas, and direct harm to nontarget organisms. Some of the major challenges for predictive risk assessment include characterizing gene flow over time and space, plant fitness in changing environments, and impacts to nontarget organisms, communities and ecosystems. The lack of baseline information about the ecology and biodiversity of urban areas, gardens, and natural ecosystems limits the ability to predict potential hazards, identify exposure pathways, and design hypothesis-driven research. The legacy of introduced ornamental plants as invasive species generates special concern about future invasions, especially for GM plants that exhibit increased stress tolerance or adaptability. While ecological risk assessments are a valuable tool and have helped harmonize regulation of GM plants, they do not define the acceptable level of risk or uncertainty. That responsibility belongs to regulators, stakeholders and citizens.

Altered development of Arabidopsis thaliana carrying the Agrobacterium tumefaciens ipt gene is partially due to ethylene effects

Transgenic Arabidopsis thaliana plants containingthe Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increasedcytokinin (CK) levels on the development of this rosette plant species. Inthreeindependently transformed lines (ipt-156, 158 and 161),Arabidopsis plants had smaller leaves, an underdevelopedroot system and decreased apical dominance in inflorescence stems. The smallertransgenic leaves were highly serrated along the margins, pale green and hadpointed leaf tips. In cross section, transgenic leaves had smaller cells andirregularly shaped epidermal cells. In the ipt-161 line,leaves and hypocotyls frequently exhibited purple color due to anthocyaninproduction. The most severe phenotype was observed in tissue cultureconditions,while growth in soil reduced or eliminated some phenotypic effects. Compared toC24 wild type plants, ipt-161 plants accumulated zeatinandzeatin riboside with an approximate 10-fold increase in the total pool of CKs.Astudy of the progeny resulting from crosses between theipt-161 transgenic line and the ethylene insensitivemutants ein1, ein2 andeti5 suggested that part of the altered developmentexhibited by the ipt transgenic plants was caused byincreased ethylene levels.

Discoveries and Dilemmas Concerning Cytokinin Metabolism.

Natural cytokinin compounds that function as plant hormones are adenine molecules connected to an N6 side chain. Metabolic pathways produce various structural modifications to the adenine moiety and/or the side chain that affect cytokinin function, activity, stability, and transport in plants. This review focuses on recent research that is elucidating the genes and biochemical pathways involved in cytokinin conjugation, deconjugation, and interconversion. One area of new discovery concerns the process of O-glucoside conjugate formation. Genes have been identified for O-glucosyltransferase enzymes in Phaseolus (ZOG1 and ZOX1) and maize (cisZOG1) and have been used to study enzyme activity, structure, and function in plant development. New information about the genetic basis of the adenine salvage pathway and its relationship to cytokinin interconversion and regulation has also become available. However, various dilemmas about cytokinin metabolism remain and need to be resolved using a combination of research approaches.

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the β-glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.

Cytokinin inhibition of Arabidopsis root growth: An examination of genotype, cytokinin activity, and N6-benzyladenine metabolism

The effects of cytokinins on the in vitro growth of the roots of Arabidopsis thaliana seedlings were examined. Root growth was inhibited in a manner dependent upon the type of cytokinin compound, the cytokinin concentration, the Arabidopsis genotype, and the duration of exposure to cytokinin. For the cytokinins N6-benzyladenine (BA), isopentenyl adenine (iP), or dihydrozeatin (DHZ), the concentration required for 50% root growth inhibition differed for each cytokinin and in each of three Arabidopsis genotypes tested. iP was the most active cytokinin in inhibiting the root growth of the Ler-0 genotype, whereas iP and BA had equal activity when tested with the Col-2 and Columbia genotypes. DHZ had the lowest activity of the three cytokinins tested in all three genotypes. A brief 1-day exposure of seeds to a root-inhibiting concentration of BA increased root growth compared with seedlings grown without BA; exposure to BA for 3–6 days inhibited root growth. BA metabolism was evaluated after 6 h and 1, 3, and 6 days in Columbia seedlings. BA, N6-benzyladenosine (BAR), and N6-benzyladenosine-5′-monophosphate (BAMP) decreased with time, whereas N6-benzyladenine-7-β-d-glucopyranoside (BA-7-G) and N6-benzyladenine-9-β-d-glucopyranoside (BA-9-G) accumulated in the growing seedlings. Seven aromatic cytokinins were compared at 5 μm for their effect on Col-3 root growth. BA, BAR, N6-(m-hydroxybenzylamino)adenine, and N6-(o-hydroxybenzylamino)adenine were highly effective in inhibiting root growth, whereas N6-(p-hydroxybenzylamino)adenine produced only a slight decrease in root growth. BA-7-G and BA-9-G did not affect root growth.

MODES OF REGENERATION IN PELARGONIUM×HORTORUM (GERANIACEAE) AND THREE CLOSELY RELATED SPECIES

SummaryModes of regeneration from hypocotyl explants were studied in Pelargonium × hortorum ‘Scarlet Orbit,’ and three wild relatives, P. zonale, P. alchemilloides, and P. inquinans, on different cytokinin treatments [1 μM thidiazuron (TDZ), 4 μM TDZ, or 8 μM N6-benzylaminopurine (BA) and 1 μM indole-3-acetic acid (IAA)]. P. × hortorum ‘Scarlet Orbit’ and P. zonale showed similar high numbers of easily detached, embryo-like structures in response to 1 μM TDZ; P. alchemilloides and P. inquinans showed weak embryogenic responses to all treatments. To revisit whether P. × hortorum produces somatic embryos, and to examine modes of regeneration in the wild species, the histology of regenerating structures on hypocotyl explants in 1 μM TDZ was examined. Both P. × hortorum and P. zonale produced embryo-like structures from single cell derivatives of epidermal cells. Globular-shaped structures transitioned into heart-shaped structures that had loose attachments to explant surfaces and no vascular connection to the explant. Roots with direct vascular connections to the rest of the embryo-like structures were never observed; root organogenesis appeared to be secondary. We propose that P. × hortorum and P. zonale exhibit partial somatic embryogenesis, in which all of the criteria for somatic embryos are met except formation of a root pole. In both species, explants forming embryo-like structures could also undergo shoot organogenesis, where shoots exhibited a broad base of attachment to the explant and a vascular connection to vascular nodules within the explant. Epidermally derived embryo-like structures were not observed in P. alchemilloides or P. inquinans in response to 1 μM TDZ. Shoot organogenesis occurred in P. alchemilloides but not in P. inquinans.

Bentgrass Distribution Surveys and Habitat Suitability Maps Support Ecological Risk Assessment in Cultural Landscapes

The bentgrasses comprise an adaptable group of grasses that include introduced species, cultivated turfgrasses, and native plants in North America. Their distribution in cultural landscapes has not been documented, and this gap in knowledge has limited the development of predictive ecological risk assessments for creeping bentgrass engineered for herbicide resistance. In this study, bentgrass distribution and abundance were surveyed in 289 plots in an 8.5 km2 site surrounding a golf course in the northeastern United States. Four introduced species and two native bentgrasses were identified in seminatural and managed plant communities. Across the study site, 77% of the plots containing creeping bentgrass also had invasive plants. Bentgrasses co-occurred with critical habitat for threatened or endangered animals. Multivariate logistic regression analysis showed that bentgrasses were positively correlated with herbaceous plant cover and mowing, but negatively correlated with tree canopy cover, shrub cover, poorly drained soils, and leaf litter. The most influential ecological factors were tree canopy cover and soil moisture. Geospatial information about these two ecological factors was combined with mathematical models to generate two habitat suitability maps. The favorable environments map (FEM) showed that highly suitable bentgrass habitat covered 36% of the study site and included common features such as home lawns and railroad right-of-ways. Our results suggest that release of herbicide-resistant creeping bentgrass in this cultural landscape could potentially result in pollen-mediated gene flow, interspecific hybridization, environmental hazards, and herbicide selection pressure in some areas. Habitat suitability maps could be critical tools for predictive ecological risk assessments, monitoring projects, and management of herbicide-resistant bentgrasses. Nomenclature: Creeping bentgrass, Agrostis stolonifera L.; AGSST

Switchgrass (Panicum virgatum L.) Genotypes Differ between Coastal Sites and Inland Road Corridors in the Northeastern US.

Switchgrass (Panicum virgatum L.) is a North American grass that exhibits vast genetic diversity across its geographic range. In the Northeastern US, local switchgrass populations were restricted to a narrow coastal zone before European settlement, but current populations inhabit inland road verges raising questions about their origin and genetics. These questions are important because switchgrass lines with novel traits are being cultivated as a biofuel feedstock, and gene flow could impact the genetic integrity and distribution of local populations. This study was designed to determine if: 1) switchgrass plants collected in the Long Island Sound Coastal Lowland coastal Level IV ecoregion represented local populations, and 2) switchgrass plants collected from road verges in the adjacent inland regions were most closely related to local coastal populations or switchgrass from other geographic regions. The study used 18 microsatellite markers to infer the genetic relationships between 122 collected switchgrass plants and a reference dataset consisting of 28 cultivars representing ecotypes, ploidy levels, and lineages from North America. Results showed that 84% of 88 plants collected in the coastal plants were most closely aligned with the Lowland tetraploid genetic pool. Among this group, 61 coastal plants were similar to, but distinct from, all Lowland tetraploid cultivars in the reference dataset leading to the designation of a genetic sub-population called the Southern New England Lowland Tetraploids. In contrast, 67% of 34 plants collected in road verges in the inland ecoregions were most similar to two Upland octoploid cultivars; only 24% of roadside plants were Lowland tetraploid. These results suggest that cryptic, non-local genotypes exist in road verges and that gene flow from biofuels plantations could contribute to further changes in switchgrass population genetics in the Northeast.