Don W. Smith

Differential estrogenic activities of male and female plant extracts from two dioecious species

Abstract The reconstituted steroid transcription unit in Saccharomyces cerevisiae transformed with both a human estrogen receptor expression plasmid (YEPE10) and a reporter plasmid (YRPE2) was used to screen for estrogen compounds in cell extracts prepared from female and male plants of osage-orange, Maclura pomifera (Raf.) Scheind. and mulberry, Morus microphylla Buckl. ( Moraceae ). Phytoestrogens in the plant extracts induced the transcription of the reporter gene in transgenic yeast. The transcriptional activity increased proportionally with increased amounts of plant extracts added to the yeast cells. Female mulberry and osage-orange plant extracts activated transcription of the steroid reporter gene about 15 times and 4 times, respectively, compared to the corresponding male plant extracts. The putative phytoestrogen from Maclura was lipid soluble, and co-migrated with sterols (17 β-estradiol) and isoflavones (genistein) in TLC separations. The active fractions recovered from TLC plates exhibited UV-absorption spectra similar to authentic estradiol and genistein. The putative phytoestrogen appeared to be synthesized at specific developmental stages in female Maclura plants; levels of transcriptional activity were higher at times prior to and during flowering (February–April). Moreover, extracts from monoecious members of Moraceae , Ficus species (fig and rubber tree) did not activate transcription of the steroid reporter gene in the yeast system. Collectively, these data correlate the occurrence and levels of endogenous phytoestrogens with female individuals of two dioecious species, suggesting a possible pattern or strategy in the reproductive ecology of these dioecious species.

Phytoestrogens and floral development in dioecious Maclura pomifera (Raf.) Schneid. and Morus rubra L. (Moraceae)

Using a sensitive and highly specific steroid-regulated transcription system in Saccharomyces cere6isiae to screen for estrogen mimetics in plant extracts, differential estrogenic activities of male and female extracts from two dioecious species were recently discovered (Maier et al., Plant Sci., 109 (1995) 31‐43). Phytoestrogens in extracts of Maclura pomifera (Raf.) Schneid. and Morus rubra L. (Moraceae) appeared to be active at specific developmental stages. The levels of b-galactosidase transcriptional activity were higher prior to and during flowering (December‐April) and during formation of new buds for the following year (July‐December). Seasonal stages of floral development were compared between male and female individuals by scanning electron microscopy. There were no rudimentary gynoecia found in the male flowers or rudimentary androecia in the female flowers of Maclura pomifera at any stage of floral development. There were no rudimentary androecia found in female flowers of Morus rubra and M. alba at any stage. However, a vestigial gynoecium was formed in the male flower just prior to anthesis. An association between high levels of transcriptional activities and the formation of functional gynoecium in female flowers of both species and vestigial gynoecium in mulberry male flowers was found. Interference-based assays with the GAL4-ERE overlapping promoter elements in the reporter plasmid of S. cere6isiae strain BJ2168 indicated that the phytoestrogens acted via the estrogen receptor in activating the transcription of the reporter gene. Together, these data raise the possibility that phytoestrogens act through endogenous receptors in regulating the expression of target genes which may influence the development of female reproductive structures.

High-performance liquid chromatography of deoxyribonucleoside di- and triphosphates in tomato roots

Abstract The levels of endogenous deoxyribonucleotides in plants are greatly affected by the absence of the micronutrient boron. Described here is a high-performance liquid chromatographic assay for quantitating deoxyribonucleoside di- and triphosphates in tomato root tip tissues under boron-sufficient and boron-deficient conditions. The extraction procedure consists of (a) removing phenols from the tomato root tips by grinding frozen tissue in 6% trichloroacetic acid containing polyvinylpolypyrrolidone, (b) neutralizing the extract with freon-amine, (c) selective degradation of ribonucleotides with periodate, followed by (d) treatment with rhamnose and methylamine prior to injection of the deoxyribonucleotides into an anion-exchange column. Results indicate that the eight deoxyribonucleoside di- and triphosphates can be reproducibly resolved and quantified. By this extraction procedure, column life was extended to more than 70 runs per cartridge. This assay technique can be used with less than 500 mg of fresh root tip tissue and allows quantitation of plant deoxyribonucleotides in the picomole range.

Nucleotide extraction and quantitation from tomato roots by high-performance liquid chromatography

Abstract The chromatographic separation and quantitation of nucleotides in tomato root tips are seriously impaired by the presence of phenols, which greatly increase in the absence of boron. However, when polyvinylpolypyrolidone is added to the 6% trichloroacetic acid homogenizing solution and the mixture is shaken on ice for 30 min, there is a decrease in phenol concentration such that nucleotides can be separated and quantified on anion-exchange Partisil SAX10 columns after neutralization with freon—amine. This treatment extends column life to more than 70 analyses per cartridge. Such a technique is unique in that it can be used with less than 500 mg of root tip tissue and allows quantitation of plant nucleotides in the picomol range.

Microscopic examination on cytological changes in Allium cepa and shift in phytoplankton population at different doses of Atrazine

Atrazine is a wide-range herbicide. For over 50 years, atrazine has been used as a selective broadleaf herbicide in many capacities, from pre-plant to pre-emergence to post-emergence, depending on the crop and application. Currently, 96% of all atrazine used is for commercial applications in fields for the control of broadleaf and grassy weeds in crops such as sorghum, corn, sugarcane, pineapple and for the control of undesirable weeds in rangeland. Many panhandle wells have also detected atrazine in samples taken. The concern for the public is the long-term effect of atrazine with its increasing popularity, and the impact on public health. We investigated the effect of different concentrations of atrazine on Allium cepa (onion), a standard plant test system. We established a control with the Allium bulbs grown on hydroponics culture. Varying concentrations of atrazine was used on the standard plant test system, Allium cepa grown hydroponically. The mitotic indices varied and with higher doses, we observed various chromosomal abnormalities including sticky bridges, early and late separations, and lag chromosomes with higher doses of treatments. In the second part of the experiment, 0.1ppb, 1ppb, 10ppb, and 100ppb concentrations of atrazine were applied to established phytoplankton cultures from the Lake Tanglewood, Texas. Study with a Sedgwick-Rafter counter, a BX-40 Olympus microscope with DP-70 camera revealed a gradual shift in the phytoplankton community from obligatory to facultative autotroph and finally to a parasitic planktonic community. This explains the periodic fish kill in the lakes after applications of atrazine in crop fields.

Characterization of some biological specimens using TEM and SEM

The advent of novel techniques using the Transmission and Scanning Electron Microscopes improved observation on various biological specimens to characterize them. We studied some biological specimens using Transmission and Scanning Electron Microscopes. We followed negative staining technique with Phosphotungstic acid using bacterial culture of Bacillus subtilis. Negative staining is very convenient technique to view the structural morphology of different samples including bacteria, phage viruses and filaments in a cell. We could observe the bacterial cell wall and flagellum very well when trapped the negative stained biofilm from bacterial culture on a TEM grid. We cut ultra thin sections from the fixed root tips of Pisum sativum (Garden pea). Root tips were pre fixed with osmium tetroxide and post fixed with uranium acetate and placed in the BEEM capsule for block making. The ultrathin sections on the grid under TEM showed the granular chromatin in the nucleus. The protein bodies and large vacuoles with the storage materials were conspicuous. We followed fixation, critical point drying and sputter coating with gold to view the tissues with SEM after placing on stubs. SEM view of the leaf surface of a dangerous weed Tragia hispida showed the surface trichomes. These trichomes when break on touching releases poisonous content causing skin irritation. The cultured tissue from in vitro culture of Albizia lebbeck, a tree revealed the regenerative structures including leaf buds and stomata on the tissue surface. SEM and TEM allow investigating the minute details characteristic morphological features that can be used for classroom teaching.