S3.1 TRANSITION TO FLOWERING AND FLOWER DEVELOPMENT IN POPULUS Amy Brunner, Rozi Mohamed, Jingyi Li, Hao Wei, Olga Shevchenko, Caiping Ma, Richard Meilan, and Steven Strauss Department of Forest Science, Oregon State University, Corvallis, OR 97331-5752 E-mail: Amy.Brunner@orst.edu The transition to flowering in poplars and other trees occurs over multiple temporal and spatial scales. This transition involves an age/size component (i.e., tree maturation that occurs after several years), a within-tree component (i.e., location in the crown and shoot), and a seasonal component. As with most temperate trees, poplars exhibit indirect flowering, where there is a period of winter dormancy between floral initiation and anthesis. Moreover, poplars are dioecious and gender determination occurs early in flower development as only reproductive organs of a single gender are initiated. Guided by work in Arabidopsis and other annual plants, we are studying poplar homologs of flowering time, meristem identity and floral organ identity genes. In addition, we are developing genomics approaches to identify candidate poplar genes involved in maturation and flowering. Recent progress will be presented, including preliminary results from DNA expression microarrays and functional studies of poplar genes related to TERMINAL FLOWER1 and FT from Arabidopsis. Our studies of floral meristem and organ identity genes include functional studies of PTLF (poplar LEAFY/ FLORICAULA homolog) in poplar transgenics that are now in their eighth growing season. In a male genotype, an antisense-PTLF construct altered floral phenotypes in several transgenic lines, ranging from bisexual flowers and female flowers to flowers showing a profusion of carpelloid and perianth cup-like organs. In contrast, none of the female antisense-PTLF transgenics exhibited changes in floral phenotype. Co-overexpression of LEAFY and PTAG2 (poplar AGAMOUS homolog) also induced bisexual flowers in a male genotype. Current work focuses on transformation of an early-flowering poplar genotype with RNAi constructs designed to suppress various floral meristem and organ identity genes.
S3.2 GENE EXPRESSION DURING ADVENTITIOUS ROOT FORMATION IN POPULUS Barry Goldfarb 1, Qian Wu 1, Carmen Lanz-Garcia 1, Andrew Groover 2, Richard Meilan 3 and Steven Strauss 3 1 - Department of Forestry, North Carolina State University, Raleigh, NC 27695-8002 USA 2 - USDA Forest Service, Institute of Forest Genetics, Davis, CA 95616 USA 3 - Department of Forest Science, Oregon State University, Corvallis OR 97331 USA E-mail: bgg@unity.ncsu.edu We are studying genes that are expressed during adventitious root formation in Populus. In one approach, we have cloned an apparent orthologue to the NAC1 gene in Arabidopsis. NACs are a large family of plant-specific transcription factors and the AtNAC1 controls lateral root formation in response to auxin (Xie et al 2000, Genes and Development 14: 3024-3036). The sequence of the Populus NAC1 is more similar to AtNAC1 than the latter is to a shoot-meristem NAC from Arabidopsis. The Populus NAC1 is also more highly expressed in roots than in stems or leaves. Further expression studies and transgenic experiments are underway to test the role of this gene in lateral and adventitious root formation. In a second approach, T-DNA gene (no promoter) and enhancer (minimal promoter) traps are being used to isolate additional rooting genes. Three hundred lines were transformed with a gene-trap construct and an additional 311 lines with an enhancer-trap construct. All lines were screened for GUS expression during root formation in vitro, with three gene-trap and 20 enhancer trap-lines testing positive for GUS early in root formation. At the time of this writing, genomic sequences adjacent to the inserted vector have been cloned using TAIL-PCR in 14 (three gene-trap and 11 enhancer-trap) of the 23 lines. We have used the sequences of five lines to search the 2X poplar genome sequence and three of the five lines have yielded contigs that are highly similar, with E values <10-50. Results of expression studies and transgenic experiments for some of these genes will be discussed. Moreover, an additional 218 gene trap and 232 enhancer trap lines have been produced and screened for GUS expression during rooting. Nineteen lines have been selected for further study.
S3.3 ACCELERATION OF FLOWERING IN SWEETGUM USING LEAFY William H. Rottmann, Lisa M. Kless, and Shujun Chang ArborGen LLC, P.O. Box 840001, Summerville, SC, 29484, USA E-mail: whrottm@arborgen.com The Arabidopsis LEAFY (LFY) gene has been demonstrated to induce early flowering in transgenic trees when it is overexpressed from the CaMV 35S promoter. This study tested the ability of LFY to accelerate flowering in sweetgum (Liquidambar styraciflua) and compared the effectiveness of the Arabidopsis ACTIN2 (ACT2) promoter with that of the 35S promoter. A companion study was performed using tobacco. Primary transformants of tobacco showed only slight acceleration of flowering with either 35S::LFY or ACT2::LFY, but most of the offspring bearing 35S::LFY showed precocious flowering, often within four weeks of germination, after fewer than ten leaves had formed. These extreme-phenotype offspring were observed from 75% of the transgenic parents, but the proportion of such offspring varied among parents. Offspring with mild acceleration of flowering were also seen. Strong phenotypes were observed for offspring of only two ACT2::LFY tobacco transformants. Additional offspring showed mild acceleration of flowering and distorted flowers, but the overall frequency of abnormal flowering was lower than seen with 35S::LFY. When 35S::LFY was tested in sweetgum, inflorescences with primarily female reproductive organs appeared in nine of 21 lines after a variable number of months of in vitro propagation and three to five months of growth in soil. Reproductive meristems developed repeatedly through the growing season and were not limited to a specific time of year. Stored pollen was applied to flowers of several plants, and fertile seeds were recovered from two genotypes. However, only one of twelve offspring was found to have inherited the LFY transgene, and this plant did not have the same phenotype as its parent. No acceleration of flowering was seen when the ACT2 promoter was used to drive expression of LFY in sweetgum.
S3.4 IDENTIFICATION OF PROTEINS PRESENT IN DOUGLAS-FIR OVULAR SECRETIONS USING PROTEOMICS Brett Poulis and Patrick von Aderkas Centre for Forest Biology, Department of Biology, University of Victoria E-mail: bpoulis@uvic.ca Douglas-fir is wind pollinated and has an engulfment mechanism that increases pollination success. The indiscriminate nature of this mechanism allows foreign organisms such as fungal spores, bacteria, and pollen from other species to enter the ovule with Douglas-fir pollen. One week prior to fertilization, an ovular secretion exudes from the nucellar tip into the micropylar canal. The origin and contents of this secretion are unknown; however, it has been suggested that the secretion may originate from either the megagametophyte or nucellus. Many questions remain in regards to what role the secretion plays. Using proteomic methodologies such as 2D gel electrophoresis and mass spectrometry, proteins present in this secretion were identified. These protein identifications provide evidence in the secretion’s ability to affect pollen development as well as providing a defensive barrier against foreign organisms.
S3.5 GIBBERELLIN METABOLISM AND SIGNALING IN TREE GROWTH AND DEVELOPMENT Victor B. Busov 1, Richard Meilan1, David W. Pearce 2, Caiping Ma 1, Stewart B. Rood 2 and Steven H. Strauss 1 1 - Department of Forest Science, Oregon State University, Corvallis, OR 97331-5752, USA. 2 - University of Lethbridge, Department of Biological Sciences, Lethbridge, Alberta, T1K 3M4, Canada. E-mail: victor.busov@orst.edu Gibberellins (GAs) a class of plant growth regulators that control stem elongation, flowering, seed germination and other processes in plants. We report the progress in our laboratory to elucidate the GA role in regulation of tree development. Taking advantage of the efficient transformation system in poplar we were able to use insertional mutagenesis to isolate genes involved in GA metabolism, and modulate GA signal transduction through insertion of dominant transgenes.We identified through activation tagging the gene encoding the major catabolic enzyme in plants GA 2-oxidase and have characterized the phenotypic and metabolic consequences of the ectopic expression of the gene. We also demonstrate similar phenotypic consequences caused by overexpression of a bean homolog and rescue by GA application.We also report diverse phenotypic alterations in transgenic poplars caused by overexpression of two Arabidopsis repressors of GA signaling (GAI and RGL1) and demonstrate association of the transgene expression level with severity of the phenotypic changes.The implications of these mutants and approaches to understanding GA role in tree development as well as their commercial importance for fiber production and as a biosafety measure will be discussed.
S3.6 ETHYLENE-INSENSITIVE BIRCH DISPLAYS IMPAIRED DORMANCY AND EARLY FLOWERING Raili Ruonala 1, Hannele Tuominen 2 and Jaakko Kangasjärvi 1 1 - Institute of Biotechnology and Dept. of Biosciences, Div. of Plant Physiology, University of Helsinki, 000 14 Helsinki, Finland 2 - UPSC, Dept. of Plant Physiology, Umeå University, 90187 Umeå, Sweden E-mail: raili.ruonala@helsinki.fi We have transformed birch (Betula pendula) with the Arabidopsis thaliana ethylene receptor gene carrying a dominant mutation (etr1-1), which confers ethylene insensitivity to the transgenic plants. Several kanamycin-resistant lines in five different birch clones were obtained and screened for ethylene insensitivity (Vahala et al., 2003). Four transgenic lines in two clonal backgrounds were selected to study the role of ethylene in growth and development of trees. Ethylene-insensitive birch lines displayed delayed senescence under natural inductive conditions, short days, during autumn. Furthermore, the transgenic trees did not enter dormancy under photoperiodic control, and did not form terminal buds as the corresponding wild-type trees. The photoperiodic control of growth cessation and bud dormancy was studied further in wild-type and transgenic trees under controlled short-day (SD) conditions. SD-induced growth cessation was similar in all trees, but after transferring back to long day (LD), the recuperation of growth of the ethylene-insensitive trees was faster when compared to the corresponding wild type. This suggests that ethylene insensitivity in trees does not interfere with perception of light regimes, but with the entry to, and the deepness of bud dormancy, processes that are traditionally believed to be under the control of abscisic acid. Ethylene is also involved in the control of the transition of birch buds from vegetative to generative state: the transgenic, ethylene-insensitive trees started forming male flowers at the age of two years or even earlier, and female flowers during the following season of growth. In birch flowering is normally initiated at the age of 5-10 years. These results suggest that in perennial plants ethylene is involved in the regulation of at least two bud-specific processes: regulation of the entry to, and the deepness of bud dormancy, and the initiation and transition to flowering, i.e., bud identity. Vahala J., Ruonala R., Keinänen M., Tuominen H. and Kangasjärvi J. (2003) Ethylene insensitivity modulates ozone-induced cell death in birch (Betula pendula). Plant Phys. accepted.
S3.7 Øystein Johnsen 1, Carl Gunnar Fossdal 1, Ruediger Baumann 2, Jørgen Mølmann 3, Ola Gram Dæhlen 4, David Clapham 5 and Tore Skrøppa 1 1 - Norwegian Forest Research Institute. Høgskoleveien 12, 1432 Ås, Norway 2 - Technische Universität München. Lehrbereich Forstgenetik. Am Hochanger 13, 85345 Freising, Germany 3 - Department of Biology, University of Tromsø. Dramsveien 201, 9037 Tromsø, Norway 4 - Biri Nursery and Seed Improvement Centre, Oppland Forest Society, 2820 Biri, Norway 5 - Department of Forest Genetics, Swedish University of Agricultural Sciences. Box 7027, S-75007 Uppsala, Sweden E-mail: oystein.johnsen@skogforsk.no Survival and competitive successes of boreal forest trees depend on a balance between exploiting the full growing season and minimising frost injury through proper timing of hardening in autumn and dehardening in spring. Our research has shown that the female parents of Norway spruce adjust these timing events in their progeny according to the prevailing temperature conditions during sexual reproduction. Reproduction in a cold environment advances bud-set and cold acclimation in the autumn and dehardening and flushing in spring, whereas a warm reproductive environment delays these progeny traits by an unknown non-Mendelian mechanism. We have performed identical crosses in combination with timed temperature treatments during shorter and longer periods from female meiosis, pollen tube growth, syngamy and embryogenesis, tested the progenies for bud-set and frost hardiness, and concluded that the effect of temperature most likely is a response to accumulated heat during embryogenesis and seed maturation. Our first attempt to look for a molecular mechanism has revealed that transcription of PHYO, PHYP and PHYN (using RealTime PCR) all show higher transcription levels in progenies reproduced under cold conditions than their full-sibs developed under warmer conditions. This result is consistent with preliminary findings that methylation of cytosine in total DNA is higher in progenies reproduced under warm conditions than their colder full-sib counterparts. If these observations are related to methylation of phytochrome genes, we may explain why progenies with a memory of a past time cold embryogenesis are more sensitive to short days than their full-sibs with a warmer embryonic history.
S3.8 REGULATION OF FLOWERING TIME IN TREES - WHAT CAN WE LEARN FROM ANNUAL MODELS? Henrik Böhlenius, Sven Eriksson, Tao Huang, Karolina Tandre, Laurence Campaa and Ove Nilsson Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 UMEÅ, SWEDEN E-mail: Ove.Nilsson@genfys.slu.se The regulation of flowering time in the annual plant model species Arabidopsis thaliana has been extensively characterized. It consists of three different developmental pathways: the day-length dependent (long-day) pathway, the gibberellin dependent pathway and the autonomous (day-length independent) pathway. In contrast, very little is known about the regulation of flowering time in perennials, such as the very late flowering trees. We are studying the functional conservation between Arabidopsis and Populus of genes involved in flowering time regulation. We have prevoíously shown that the flower-meristem-identity gene LEAFY can be used to induce early flowering in transgenic trees. However, only single terminal flowers were produced and the flowers produced little pollen. Our ultimate goal is to produce early flowering trees that can be used as tools for a dramatically enhanced conventional breeding. We have now shown that an alternative approach, targeting genes normally involved in flowering time regulation, can induce extremely early flowering in Populus. With this approach, inflorescences with normal flowers can be induced. We are also exploring the use of inducible systems to produce a physiologically normal flowering. These various strategies will be discussed. The use of a new Populus cDNA micro array containing 20 000 different genes derived from 100 000 EST sequences, including sequences from flower-related libraries, will be discussed. This array will allow us to identify new Populus genes involved in the regulation of the juvenility-to-maturity transition. Finally, we will describe how our work characterising the regulation of flowering time in Arabidopsis during growth in non-inductive conditions can shed new light on the regulation of the reproductive transition in trees.
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