N-succinimidyl methoxyphenylacetic acid ester, an amine-directed chiral derivatizing reagent suitable for enzymatic scale resolutions

A chiral derivatizing reagent, N-succinimidyl-2-(S)-methoxy-2-phenylacetic acid ester (SMPA), directed toward reaction with primary amine-containing compounds has been synthesized and characterized. This reagent is suitable for HPLC resolution from enzymatic-scale reactions where only microgram quantities of chiral products may be obtainable. SMPA derivatization was shown to be effective in the resolution of the enantiomers of a number of different racemic compounds. SMPA was used to resolve the diastereoisomeric derivatives of a previously unknown enzymatically oxygenated product, allowing determination of the stereochemical course of the enzymatic reaction. SMPA is easily prepared from an inexpensive, commercially available, and enantiomerically pure precursor with the formation of a shelf-stable crystalline product which is utilizable in water-containing solutions. In addition to its usefulness for micro-determinations, SMPA is useful for preparative-scale resolutions of enantiomers since the reagent is cleaved from the diastereoisomeric derivative by acid hydrolysis.     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Enhanced Growth and Activity of a Biocontrol Bacterium Genetically Engineered To Utilize Salicylate

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 μg/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 × 10⁵ CFU/g to approximately 3 × 10⁸ CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 × 10⁶ CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 μg of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10⁸ CFU/g of soil by 48 h of incubation, with the fastest increase at 100 μg/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 μg/g; at 1,000 μg/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 μg of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.     

Half-lives of oat mRNAs in vivo and in a polysome-based in-vitro system

We have used a cell-free polysome-based in-vitro mRNA-degradation system to investigate the halflives of plant cell mRNAs. In order to establish the fidelity of the in-vitro system, we used cordycepin to determine the in-vivo half-lives of -tubulin and actin mRNAs in the primary leaves of 4-d-old etiolated oat (Avena sativa L.) seedlings. The in-vitro rank order of half-lives for phytochrome A (45 min), -tubulin (105 min), and actin (220 min) mRNAs mimicked the in-vivo rank order. A pulse of red light given to excised etiolated primary leaves caused an in-vivo reduction in the half-life of -tubulin mRNA. The selectivity of the polysome-based system was further demonstrated by the decrease in the half-life of -tubulin mRNA (from 105 min to 60 min) induced by a pulse of red light given to the etiolated oat seedlings prior to isolation of polysomes. Red light did not affect the apparent half-lives of phytochrome A or actin mRNAs.Abbreviations cab gene for chlorophyll-a/b-binding protein - kb(p) kilobase (pair) - phyA gene for type-I phytochrome protein - rbcS gene for ribulose-1,5-bisphosphate-carboxylase small-subunit We thank Dr. Richard B. Meagher for the pSAc3 actin clone. We thank Dr. Cecil Stewart for the use of his density-gradient fractionator, and Dr. Virginia Crane for instruction in using the fractionator. We also appreciate the helpful comments provided by the other members of the laboratory during the course of this research: Dr. Isaac John, Dr. Iffat Rahim, Linda Barnes, Bruce Held, David Higgs, and Theresa Tirimanne. This work was supported by USDA grants CRGO 88-37261-4196 and 91-37304-6397, and the Iowa State University Biotechnology Program.     

Leaf vasculature in sugarcane (Saccharum officinarum L.)

The vascular system of the leaves of Saccharum officinarum L. is composed in part of a system of longitudinal strands that in any given transverse section may be divided into three types of bundle according to size and structure: small, intermediate, and large. Virtually all of the longitudinal strands intergrade, however, from one type bundle to another. For example, virutually all of the strands having large bundle anatomy appear distally in the blade as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. These large bundles, together with the intermediates that arise midway between them, extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of bundles at the base of the blade, both the total and mean cross-sectional areas (measured with a digitizer from electron micrographs) of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Red Light-Independent Instability of Oat Phytochrome mRNA in Vivo

Phytochrome A (phyA) mRNA abundance decreased rapidly in total RNA samples isolated from 4-day-old etiolated oat seedlings following a red light pulse. Putative in vivo phyA mRNA degradation products were detectable both before and after red light treatment. Cordycepin-treated coleoptiles were unable to accumulate the chlorophyll a/b-binding protein mRNA in response to red light, indicating that cordycepin effectively inhibited mRNA synthesis. In cordycepin-treated coleoptiles, phyA mRNA rapidly decreased in abundance, consistent with the hypothesis that phyA mRNA is inherently unstable, rather than being destabilized after red light treatment of etiolated oat seedlings.     

An mRNA that specifically accumulates in maize roots delineates a novel subset of developing cortical cells

A near full-length cDNA clone (pZRP3) corresponding to an mRNA that accumulates specifically in roots of maize was isolated. The ZRP3 mRNA is ca. 600 nucleotides in length. The amino acid sequence of the predicted polypeptide is rich in leucine (16%), proline (11%), and cysteine (8.5%). The zrp3 gene appears to be expressed exclusively in roots, whereas other ZRP3-related genes are expressed in additional organs of the maize plant. In situ hybridization shows that ZRP3 mRNA accumulation is largely confined to the cells of the cortical ground meristem. Furthermore, accumulation of this mRNA occurs within a distinct subset of cortical cells, the inner three to four cell layers.Journal paper number J-14572 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project Number 2997.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation