High-Throughput Phenotyping in Plant Stress Response: Methods and Potential Applications to Polyamine Field.

High-throughput phenotyping has opened whole new perspectives for crop improvement and better understanding of quantitative traits in plants. Generation of loss-of-function and gain-of-function plant mutants requires processing and imaging a large number of plants in order to determine unknown gene functions and phenotypic changes generated by genetic modifications or selection of new traits. The use of phenomics for the evaluation of transgenic lines contributed significantly to the identification of plants more tolerant to biotic/abiotic stresses and furthermore, helped in the identification of unknown gene functions. In this chapter we describe the High-throughput phenotyping (HTP) platform working in our facility, drawing the general protocol and showing some examples of data obtainable from the platform. Tomato transgenic plants over-expressing the arginine decarboxylase 2 gene, which is involved in the polyamine biosynthetic pathway, were analyzed through our HTP facility for their tolerance to abiotic stress and significant differences in water content and ability to recover after drought stress where highlighted. This demonstrates the applicability of this methodology to the plant polyamine field.

Internet availability of prescription pharmaceuticals to the public.

BACKGROUND: The Internet holds great but uncertain promise for increased access and cost control in health care. OBJECTIVE: To determine access to and cost of prescription pharmaceuticals over the Internet. DESIGN: An Internet search conducted during February and March 1999. SETTING: The Philadelphia region. MEASUREMENTS: Data were collected on availability and cost of medications and physician Internet visits, requirements for physician prescriptions, and geographic location of Web-based companies and consulting physicians. Costs of comparable physician visits were obtained from Medicare and managed care organizations in the Philadelphia region. Costs of medications came from five Philadelphia community pharmacies. RESULTS: Forty-six Web sites were identified. Thirty-seven sites (33 based in the United States and 4 based outside the United States) required a prescription from a personal physician or from an Internet physician consultation. Nine sites based outside of the United States did not require a prescription or physician consultation. The median cost of an Internet physician visit was $70 (range, $20 to $90), more than 15% higher than that for a general practice visit in the Philadelphia region. Quality of physician consultation, physician qualifications and specialty, and geographic location were unknown. Median price per pill of the two most commonly offered medications was 10% higher on the Internet (before shipping charges) than at Philadelphia pharmacies ($5.49 and $4.50 for sildenafil; $1.94 and $1.83 for finasteride). CONCLUSIONS: The Internet may expand patient access to health-related services but at overall increased cost. In addition, the quality of physician Internet care is uncertain, and potential for serious abuse exists. Patients can easily provide incorrect or false information to obtain medications. Furthermore, conflict of interest exists for Web-based firms because they profit from selling medications and physician consultations.

Plant low-molecular-weight phospholipase A2S (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa).

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A2 (PLA2) from developing elm seed endosperm. This represented the first purified and characterized PLA2 from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 1 19 amino acids (PLA2-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA2-II. preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA2. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca(2+)-binding loop and active-site motif, which are characteristic of animal secretory PLA2s. A soluble PLA2s activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA2s. The purified rice PLA2 consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA2-I and PLA2-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA2s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.

Specific sequence modifications of a cry3B endotoxin gene result in high levels of expression and insect resistance.

Solanum melongena (eggplant) cv. Picentia and the wild species Solanum integrifolium were transformed with both a wild type (wt) and four mutagenized versions of Bacillus thuringiensis (Bt) gene Bt43 belonging to the cry3 class. The Bt gene was partly modified in its nucleotide sequence by replacing four target regions (W: +1 to +170; X: +592 to +1057; Y: +1203 to +1376; Z: +1376 to +1984) with synthetic fragments obtained by polymerase chain reaction amplification of crude oligonucleotides. The synthetic Bt genes were designed to avoid, in their modified regions, sequences such as ATTTA sequence, polyadenylation sequences and splicing sites, which might destabilize the messenger RNA. Furthermore, the codon usage was improved for a better expression in the plant system. The amino acid composition was not altered. Four versions of the modified Bt gene were obtained, BtE, BtF, BtH and BtI, with a nucleotide subtitution percentage of 8.2, 8.6, 14, and 16%, respectively, in comparison to the wt gene Bt43. Modified versions contained different subsets of substituted regions: BtE-W + Z, BtF - Y + Z, BtH-X + Y + Z, BtI - W + X + Y + Z. In the final modified version (BtI), overall guanine+cytosine was increased from the 34.1% of the wt gene to 45.5%, and most of the destabilizing sequences were eliminated. Transgenic plants obtained with the more modified versions, BtH and BtI, were fully resistant to Leptinotarsa decemlineata Say first- and third-instar larvae, while Bt43 wt, BtE and BtF genotypes did not cause mortality and did not affect larval development.

Functional analysis of the sporulation-specific SPR6 gene of Saccharomyces cerevisiae.

The SPR6 gene of Saccharomyces cerevisiae encodes a moderately abundant RNA that is present at high levels only during sporulation. The gene contains a long open reading frame that could encode a hydrophilic protein approximately 21 kDa in size. This protein is probably produced by the yeast, because the lacZ gene of Escherichia coli is expressed during sporulation when fused to SPR6 in the expected reading frame. SPR6 is inessential for sporulation; mutants that lack SPR6 activity sporulate normally and produce viable ascospores. Nonetheless, the SPR6 gene encodes a function that is relevant to sporulating cells; the wild-type allele can enhance sporulation in strains that are defective for several SPR functions. SPR6 is located on chromosome V, 14.4 centimorgans centromere-distal to MET6.