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Carol M. Foster and William R. Graves

Desiccation damage in ornamental plant species is of particular concern to the nursery and landscape industry. Species in two genera, Acer and Alnus, display fundamental differences in how drought affects leaves. The same soil moisture content that causes foliar desiccation and abscission in Alnus maritima (Marsh.) Nutt. (seaside alder) causes neither response in Acer rubrum L. (red maple). Understanding molecular mechanisms associated with plant response to drought stress can be an im portant factor in developing strategies for improved sustainability in urban landscapes. Our objective was to characterize expression of drought-induced dehydrin genes in leaves of `Red Sunset' red maple (desiccation-resistant) and seaside alder (desiccation-sensitive) in response to dehydration and rehydration. Potted cuttings grown in a glasshouse were subjected to four cycles of drought and rehydration. Stomatal conductance and volumetric moisture content of rooting medium were used to determine when drought cycles ended. During the second and fourth cycles, leaves were sampled for RNA and protein extraction. Dehydrin probes were generated from genomic DNA of both species by using PCR with primers designed from conserved regions in dehydrin genes. Southern blot analyses revealed the presence of dehydrin genes in seaside alder and red maple genomes. Reverse transcriptase (RT)-PCR was used to isolate desiccation-induced dehydrin cDNAs from total RNA extracted from drought-stressed leaves. The cDNA clones show 61% to 66% identity at the nucleic acid level with dehydrin genes of soybean, sunflower, radish, and potato. Accumulation of dehydrin transcripts and proteins in leaves in response to dehydration and rehydration are being studied through northern and western blot analyses, respectively. Our results may lead to a rapid screening technique for seedlings with improved mechanisms of drought resistance.

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Harry T. Horner, David J. Hannapel, William R. Graves, Carol M. Foster, David J. Hannapel, William R. Graves, Carol M. Foster, Harry T. Horner and Carol M. Foster

Early nodulin genes, such as ENOD2, play a role in the first stages of nodulation. Although ENOD2 is conserved among nodulating legumes studied to date, its occurrence and activity have not been studied among woody legumes such as Maackia amurensis Rupr. & Maxim. Our objective was to localize MaENOD2 transcripts during nodule development and describe the anatomy of nodules formed on the roots of M. amurensis in relation to ENOD2 mRNA accumulation. Nodules (<1 mm, 1-2 mm, >2 mm in diameter, and mature) were prepared for light microscopy, sectioned, and stained with safranin and fast green for structural contrast or with the periodic acid Schiff's reaction for starch. The location of ENOD2 transcripts was determined by using in situ hybridization with DIG-labeled sense and antisense RNAs transcribed from a 602-bp fragment of the coding region of MaENOD2. Mature nodules from M. amurensis possessed peripheral tissues, a distal meristem, and a central infected region characteristic of indeterminant development. In situ hybridization showed that MaENOD2 transcripts accumulated in the distribution layer and uninfected cells of the central symbiotic region. Amyloplasts that contained starch grains were identified in these tissues and in the inner parenchyma of the nodule. Throughout nodule development, transcripts were restricted to areas with high levels of stored starch that surrounded cells actively fixing N2. Our results suggest that ENOD2 in M. amurensis may be a cell wall component of tissues that regulate nutrient flow to and from sinks, such as symbiotic regions of a nodule. These data may lead to a better understanding of the role of the ENOD2 gene family during nodulation.

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Carol M. Foster, William R. Graves and Harry T. Horner

Early nodulin genes, such as ENOD2, may be conserved and could function as molecular markers for nodulation. Many nodulating and nonnodulating legumes must be analyzed before the role of such genes in nodulation can be determined. Japanese pagodatree and American yellowwood are closely related, ornamental woody legumes. Unsubstantiated reports of nodulation in Japanese pagodatree require confirmation, and American yellowwood has not been observed to nodulate. We investigated the presence of putative ENOD2 genes in these species, and we are studying differential and temporal expression. Genomic DNA of Japanese pagodatree and primers, derived from proline-rich pentapeptide repeats of conserved ENOD2 sequences, were used to obtain a 555-bp PCR fragment. This cloned fragment was used as a probe for Southern and Northern hybridizations. Genomes of Japanese pagodatree and American yellowwood contained sequences that are similar to ENOD2 sequences in other legumes. Treatments with either cytokinin or an auxin transport inhibitor may induce expression of the putative ENOD2 genes. New data on the characteristics of nodulin genes in woody legumes will clarify the nature and evolution of nodulation in legumes and may have implications for developing sustainable nursery production protocols.

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Carol M. Foster, William R. Graves and Harry T. Horner

A complete picture of legume nodulation has yet to be elucidated. Most studies of the molecular mechanisms responsible for nodule organogenesis have focused on herbaceous legumes. We investigated the presence of a putative ENOD2 gene and studied the temporal and organ-specific production of its transcripts in an ornamental woody legume, Amur maackia. Primers derived from proline-rich pentapeptide repeats of conserved ENOD2 sequences and the genomic DNA of Amur maackia were used to obtain a 543-bp PCR fragment. Southern and Northern blots were probed with this cloned fragment. The Amur maackia genome contained an ENOD2 sequence that is similar to sequences in other species. Expression of the putative ENOD2 gene was detected in roots, 4 days after rhizobial inoculation, but not in leaves or stems. New data on the characteristics of nodulin genes in woody legumes will be beneficial in clarifying the nature and evolution of nodulation in legumes and may have implications for developing sustainable nursery production protocols.

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Carol M. Foster, William R. Graves and Harry T. Horner

Knowing whether leguminous trees have the potential to nodulate after infection by rhizobial bacteria is important for managing nitrogen (N) applications during tree production and for culture in the landscape. Although 98% of studied species in the Papilionoideae nodulate, the nodulation status of two tree species in this subfamily is uncertain. Cladrastis kentukea (Dum.-Cours.) Rudd (American yellowwood) did not form nodules during inoculation studies in 1939 and 1992. Nodules were observed on mature Sophora japonica L. (Japanese pagodatree) in Japan and Hawaii in the 1940s, but compatible rhizobia reportedly isolated in Japan are no longer held in bacterial collections. Our objective was to verify further that American yellowwood does not nodulate and to confirm reports that Japanese pagodatree does nodulate. Rhizobia that infect many plant hosts, soil samples and rhizobial isolates from other Sophora spp., and soil samples from mature American yellowwood and Japanese pagodatree were used to inoculate 5-day-old seedlings of American yellowwood, Japanese pagodatree, and control species. Soil from indigenous and introduced trees in the continental United States, Hawaii, Japan, and China was used. Inoculated and uninoculated plants were grown for 7 weeks in sterile Leonard jars or clay pots containing perlite and irrigated with sterile, N-free Hoagland's solution. No inoculation treatment elicited nodulation of American yellowwood or Japanese pagodatree. Our results provide additional evidence that American yellowwood lacks that capacity to nodulate and cast further doubt on nodulation of Japanese pagodatree.

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David W. Ramming, Richard L. Emershad and Carol Foster

Various in vitro conditions for culture of ovules prior to extraction and culture of immature embryos of peach [Prunus persica (L.) Batsch] and nectarine [Prunus persica (L.) Batsch var. nucipersica Schneid.] were investigated. Culture vessels consisting of test tubes, petri dishes, and polycarbonate jars were tested along with various types of support and nutrient media. Agar support was superior to liquid media with filter paper supports. Agar produced the largest embryos with 90% to 93% being converted into plants compared to liquid with only 1% to 12% embryo conversion. The best ovule orientation and support was with the micropyle down and pushed halfway into an agar-gelled medium. In experiments two and three, test tubes with vertical ovule orientation (micropyle end of ovule pushed into agar) produced larger embryos, the largest plants and the greatest percentage of embryos that converted into plants (60% and 91%). Petri dish treatments were less successful in embryo conversion than test tubes and polycarbonate jars. The addition of activated charcoal (AC) to an agar-gelled medium produced significantly larger embryos with a similar conversion rate. The addition of an agar-gelled medium to culture vessels reduces preparation time compared to filter paper supports, and placing each ovule within a test tube eliminates cross contamination, making immature embryo culture more successful.

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Carol M. Foster, William R. Graves and Harry T. Horner

ENOD2 and other early nodulin genes are conserved among legumes studied to date and might function as markers for the potential of legumes to nodulate. Early nodulin genes have been characterized only among herbaceous legumes. We are interested in understanding the nature of ENOD2 in a nodulating, woody legume. A 561-bp MaENOD2 PCR fragment was used as a probe to screen a cDNA library from nodules ≈1 mm in diameter on roots of Amur maackia, the only temperate and horticulturally desirable leguminous tree species known to nodulate. Five cDNAs were selected for nucleotide sequence analysis. Sequences were determined by using automated dideoxy sequencing and analyzed for identity to other genes with the Genetics Computer Group (GCG) program. The cDNA clones show 68% to 74% identity at the nucleic acid level with ENOD2 genes of Sesbania rostrata Brem. & Oberm., Glycine max (L.) Merrill, and Lupinus luteus L. Southern and northern analyses are being conducted to investigate the possibility of a gene family and to show differential and temporal production of transcripts, respectively. These studies provide new information about nodulins of woody legumes and are being used to facilitate related research on molecular barriers to nodulation in the closely related, non-nodulating tree species Cladrastis kentukea (Dum.-Cours.) Rudd (American yellowwood) and Sophora japonica L. (Japanese pagodatree).