Gary M. Banowetz

Dehydrin expression and drought tolerance in seven wheat cultivars

rarely receives additional moisture during emergence. The lack of precipitation during seedling emergence The winter wheat (Triticum aestivum L.) producing region of the represents a major cropping risk to producers. ConseU.S. Pacific Northwest (PNW) is subject to periods of water deficit during sowing and grain filling. Improving the genetic adaptation of quently, there is need to improve the genetic tolerance wheat to drought stress represents one of the main objectives of of wheat to drought at the seedling stage. regional breeding programs. One biochemical response to dehydrative Plant breeding efforts to improve drought tolerance stress is the accumulation of a family of proteins called dehydrins, would be aided by the identification of biochemical which are believed to protect membranes and macromolecules against markers associated with improved field performance denaturation. Although previous studies demonstrated the accumulaunder drought conditions. Dehydrins, also known as tion of dehydrins in drought-stressed wheat, little was known about late embryogenesis abundant (LEA) D11 (Dure, 1993) the relation of dehydrin expression to acquisition of drought tolerance proteins represent potential markers. Dehydrins are in specific varieties adapted to the PNW. We characterized dehydrin members of a family of proteins that are expressed after accumulation during the exposure of seven cultivars (‘Connie’, ‘Gene’, ‘TAM105’, ‘Rod’, ‘Hiller’, ‘Rhode’, and ‘Stephens’) to progressive plants are exposed to stresses with a dehydrative compodrought stress in four separate experiments. The objective was to nent. This family of proteins is characterized by the identify differences in the nature or timing of dehydrin expression in presence of a consensus amino acid sequence (EKK these cultivars and to learn whether dehydrin expression was associGIMDKIKELPG) near the carboxy terminus (Close ated with the acquisition of stress tolerance during seedling developet al., 1993). Dehydrins can be detected by means of ment. Expression of a 24-kDa dehydrin was observed in Connie, antibodies prepared against this consensus sequence TAM105, and Gene after 4 d of stress and at subsequent sampling (Close et al., 1993) and have been identified in at least dates while no dehydrins were detected in nonstress control plants. 30 diverse plant species including wheat (Campbell and Dehydrin expression was significantly delayed in the remaining cultiClose, 1997). vars. The presence of this dehydrin was related to acquisition of drought tolerance characterized by a greater maintenance of shoot An association between tolerance to stresses with a dry matter production in Connie, TAM105, and Gene. Although the dehydrative component (drought, freezing, or salinity) role of these proteins remains unknown, their association with stress and the expression of dehydrin proteins has been obtolerance suggests that dehydrins might be used to improve the adaptaserved in some crop species. Houde et al. (1992) found tion to drought. that the expression of a specific dehydrin (WSC120) accompanied the development of freezing tolerance in eight species of Gramineae. Tolerance to chilling temperatures during emergence was correlated with the M wheat-producing regions of the world are expression of a 35-kDa dehydrin in two genetically simisubject to water deficits during some part of the lar cowpea [Vigna ungiculata (L.) Walp] sublines that growing season (Moustafa et al., 1996). The impacts of differed in their expression of this dehydrin (Ismail et al., these water deficits on grain development and yield 1997). Lim et al. (1999) also found a positive association depend on their severity and the stage of plant growth between cold hardiness and a dehydrin protein in Rhoduring which they occur. Seedling emergence is one dodendron. Danyluk et al. (1998) showed that the stage of growth that is sensitive to water deficit. In WCOR410 dehydrin protein accumulated near the Mediterranean environments like the PNW, dry condiplasma membrane during cold acclimation of wheat and tions during emergence and early growth along with low suggested that this accumulation protected the integrity temperatures during winter and high temperatures and of the plasma membrane when plants were subjected increasing water demands at the end of spring, result to stress. Zhu et al. (2000) reported increased expression in low yields because of the inability of plants to produce of dehydrin genes during the development of freezing adequate dry matter (Regan et al., 1992). Many productolerance in a more tolerant barley (Hordeum vulgare ing regions of the world, including the PNW are subL.) cultivar Dicktoo relative to that which occurred in jected to water deficits during the seedling stage since ‘Morex’, a less tolerant variety. Cellier et al. (1998), winter wheat is sown during autumn into dry soil and using two sunflower (Helianthus annuus L.) inbred lines, one tolerant and one susceptible to drought stress, showed a higher accumulation of mRNA transcripts Cesar G. Lopez, Catedra de Mejoramiento Vegetal, Facultad de Ciencias Agrarias, Universidad Nacional de Lomas de Zamora, Ruta 4 corresponding to HaDhn1 and HaDhn2 genes in the Km. 2, Llavallol, 1832, Buenos Aires, Argentina; Gary M. Banowetz, tolerant line that was associated with cellular turgor USDA/ARS, 3450 S.W. Campus Way, Corvallis, OR 97331; C. James maintenance under drought stress. Peterson, Department of Crop and Soil Science, Oregon State UniverAlthough genotypic differences in dehydrin expressity, Corvallis, OR 97331-3002; Warren E. Kronstad (deceased), Department of Crop and Soil Science, Oregon State University, Corvallis, sion have been demonstrated in responses to cold and OR 97331. Received 8 July 2002. *Corresponding author banowetg@ drought tolerance, it is important to study the expression ucs.orst.edu). of dehydrins in relation to changes in leaf water potential when seedlings are exposed to drought. The purpose Published in Crop Sci. 43:577–582 (2003).

A hybrid genetic algorithm for multiobjective problems with activity analysis-based local search

The objective of this research was the development of a method that integrated an activity analysis model of profits from production with a biophysical model, and included the capacity for optimization over multiple objectives. We specified a hybrid genetic algorithm using activity analysis as a local search method, and NSGA-II for calculation of the multiple objective Pareto optimal set. We describe a parallel computing approach to computation of the genetic algorithm, and apply the algorithm to evaluation of an input tax to regulate pollution from agricultural production.

An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6

BackgroundPseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species.ResultsWe used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system.ConclusionsThe improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.

4-Formylaminooxyvinylglycine, an herbicidal germination-arrest factor from Pseudomonas rhizosphere bacteria.

A new oxyvinylglycine has been identified as a naturally occurring herbicide that irreversibly arrests germination of the seeds of grassy weeds, such as annual bluegrass (Poa annua), without significantly affecting the growth of established grass seedlings and mature plants or germination of the seeds of broadleaf plant species (dicots). Previously, Pseudomonas fluorescens WH6 and over 20 other rhizosphere bacteria were isolated and selected for their ability to arrest germination of P. annua seeds. The germination-arrest factor (GAF, 1) responsible for this developmentally specific herbicidal action has now been isolated from the culture filtrate of P. fluorescens WH6. Purification of this highly polar, low molecular weight natural product allowed its structure to be assigned as 4-formylaminooxy-l-vinylglycine on the basis of NMR spectroscopic and mass spectrometric data, in combination with D/L-amino acid oxidase reactions to establish the absolute configuration. Assay results for P. annua inhibition by related compounds known to regulate plant growth are presented, and a cellular target for 1 is proposed. Furthermore, using bioassays, TLC, and capillary NMR spectroscopy, it has been shown that GAF (1) is secreted by all other herbicidally active rhizosphere bacteria in our collection.

High resolution characterization of soil biological communities by nucleic acid and fatty acid analyses

Fatty acid methyl ester (FAME) and length heterogeneity-polymerase chain reaction (LH-PCR) analyses were used to generate ‘fingerprints’ of FAMEs and eubacterial 16S rDNA sequences characteristic of agricultural soil communities. We hypothesized that pooling data from two methods that characterized different components of soil biological communities would improve the resolution of fingerprints characterizing the effects of contrasting tillage and ground cover practices. By using supervised classifications of FAME and LH-PCR, a discriminant analysis procedure distinguished soils from contrasting tillage and ground cover management and predicted the origin of soil samples. Used independently, FAME provided higher resolution of tillage, ground cover, and field location than LH-PCR, but LH-PCR was effective at identifying field location. Pooling data from both methods did not enhance the predictive power. A comparison of linear discriminant analysis, quadratic discriminant analysis, and nonparametric density estimation demonstrated that minimizing assumptions about data distribution improved the capacity of FAME analysis to resolve differences in soil types. Use of a purely statistical Bayesian method to select a subset of fatty acids (FA’s) as variables in discriminant analyses identified FA’s that represented signature FA’s for specific groups of organisms. Published by Elsevier Science Ltd.

Impact of land use patterns and agricultural practices on water quality in the Calapooia River Basin of western Oregon

Agricultural practices, including tillage, fertilization, and residue management, can affect surface runoff, soil erosion, and nutrient cycling. These processes, in turn, may adversely affect (1) quality of aquatic resources as habitat for amphibians, fish, and invertebrates, (2) costs of treating surface and ground water to meet drinking water standards, and (3) large-scale biogeochemistry. This study characterized the surface water sources of nitrogen (N) (total, nitrate [NO3−], ammonium [NH4+], and dissolved organic N) and sediment active within 40 subbasins of the Calapooia River Basin in western Oregon in monthly samples over three cropping years. The subbasins included both independent and nested drainages, with wide ranges in tree cover, agricultural practices, slopes, and soils. Sediment and N form concentrations were tested against weather and agricultural practice variables. Subbasin land use ranged from 96% forest to 100% agriculture. Average slopes varied from 1.3% to 18.9%, and surface water quality ranged from 0.5 to 43 mg L−1 (ppm) total N maxima and 29 to 249 mg L−1 suspended sediment maxima. Total N during the winter was positively related to percentage landcover of seven common agricultural crops (nongrass seed summer annuals, established seed crops of perennial ryegrass [Lolium perenne L.], tall fescue [Schedonorus phoenix {Scop.} Holub], orchardgrass [Dactylis glomerata L.], clover [Trifolium spp.], and newly planted stands of perennial ryegrass and clover) and negatively related to cover by trees and one seed crop, Italian (annual) ryegrass (Lolium multiflorum). Results for NO3− and total N were highly similar. Sediment concentrations were most strongly related to rainfall totals during periods of 4 and 14 days prior to sampling, with smaller effects of soil disturbance. Fourier analysis of total N over time identified four prominent groups of subbasins: those with (1) low, (2) medium, and (3) high impacts of N (up to 2, 8, and 21 mg L−1, respectively) and a strong cyclical signal peaking in December and (4) those with very high impact of N (up to 43 mg L−1) and a weak time series signal. Preponderance of N in streams draining agriculturally dominated subbasins was in the form of the NO3− ion, implying mineralization of N that had been incorporated within plant tissue following its initial application in the spring as urea-based fertilizer. Since mineralization is driven by seasonal rainfall and temperature patterns, changes in agronomic practices designed to reduce prompt runoff of fertilizer are unlikely to achieve to more than ~24% reduction in N export to streams.

Hormones in wheat kernels during embryony

Summary Abscisic acid (ABA), indole-3-acetic acid (IAA) and six cytokinins in wheat (Triticum aestivum L.) grain components were extracted, from 0 to 25 d post anthesis (DPA), and quantified by noncompetitive indirect ELISA. At 3 DPA, which coincides with early embryo and endosperm cell divisions, whole kernel hormone levels in µmol kg –1 dry mass (DM) were: zeatin (Z) 30, IAA 4, zeatin riboside 1.8, ABA 1.0, other cytokinins < 0.6. Endosperm Z content declined from about 13 µmol kg –1 DM at 6 DPA to 0.1µmol kg –1 DM by 9 DPA and remained low thereafter. Generally by 9 DPA, embryo differentiation is nearly complete, and the endosperm mitotic index has decreased to near zero. At 13 DPA, Z content in embryos was 0.9 µmol kg –1 DM, and then decreased to 0.3 µmol kg –1 DM by 25 DPA. Endosperm IAA content remained low through 9 DPA (about 6 µmol kg –1 DM) and then increased sharply to about 38 µmol kg –1 DM by 19 and 25 DPA. In contrast, embryo IAA content decreased from about 34 µmol kg –1 DM at 13 DPA to about 15 µmol kg –1 DM at 19 and 25 DPA. Endosperm ABA content fluctuated between 1.0 and 4.5 µmol kg –1 DM between 6 and 25 DPA while embryo ABA content fluctuated between 3.4 and 4.8 µmol kg –1 DM between 13 and 25 DPA. These hormone changes may be

Conservation Practices in Western Oregon Perennial Grass Seed Systems

Rapid changes in practices used to produce perennial grass seed crops in the U.S. Pacific Northwest region and shortened lengths of time that perennial grass seed fields remain in production have increased the need for additional rotation crops that are adapted to the poorly drained soils found in western Oregon. This research was conducted at three sites to determine ways to manage meadowfoam (Limnanthes alba Hartw. ex Benth.) as a component in perennial grass seed rotation systems. Experiments were conducted in 1997, 1998, and 2001 to investigate combinations of spring-applied herbicide and N fertilizer and times of applications, direct-seeded and conventional tillage establishment methods, and previous crop residue management on meadowfoam seed yield, seed oil concentration, and oil yield. No spring-applied fertilizer or herbicide produced responses for all yield components as great as or greater than any other treatment combination. Direct-seeded meadowfoam yielded more oil than the conventional establishment treatment. There was no effect of residue management amounts from grass seed grown in the previous rotation sequence on meadowfoam production; however, maximal residue management, especially if used in combination with direct-seeded meadowfoam, should reduce annual soil erosion. Meadowfoam is suited to low-input production and is adapted to the use of conservation practices including direct seeding and maximal residue management in perennial grass seed systems.

Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P. aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E. amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P. fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E. amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.