Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanold synthesis. The role of PAL In pathway regulation was investigated by measurement of product accumulation as a function of enzyme activity in a coilection of near-isogenic transgenic tobacco plants exhibiting a range of PAL levels from wild type to 0.2% of wild type. In leaf tissue, PAL level is the dominant factor regulating accumulation ofthe major product chlorogenic acid and overall flux into the pathway. In stems, PAL at wild-type levels contributes, together with downstream steps, in the regulation of lguin deposition and becomes the dominant, rate-determining step at levels 3-to 4-fold below wild type. The metabolic impact of elevated PAL levels was investigated in transgenic leaf callus that overexpressed PAL. Accumulation of the flavonoid rutin, the major product in wild-type callus, was not increased, but several other products accumulated to similarly high levels. These data indicate that PAL is a key step in the regulation of overall flux into the pathway and, hence, accumulation of major phenylpropanoid products, with the regulatory architecture of the pathway poised so that downstream steps control partitioning into different branch pathways.

An in vivo shikimate accumulation assay with excised leaf tissue was developed to provide a fast and reliable method for identifying glyphosate-resistant plants. The assay is based on glyphosate-induced accumulation of shikimate. There was a linear accumulation of shikimate in excised leaf discs of soybean and canola treated with 250 μM glyphosate for 48 h. The IC50 for the accumulation of shikimate in soybean and corn leaf discs was 34 and 87 μM, respectively. Leaf discs excised from glyphosate-resistant corn or soybean did not accumulate shikimate when treated with 500 μM glyphosate. Leaf discs taken from a number of field-grown plants accumulated shikimate in a glyphosate dose–dependent manner. The accumulation of shikimate was dependent on light and the age of the leaf from which the disc was taken. The assay worked either in 96-well microtiter plates or in vials, and it clearly differentiated between glyphosate-resistant and -susceptible crops in which the resistance is due to an alteration of the target site for glyphosate. The assay was simple and robust and has the potential to be used as a high throughput assay to detect glyphosate resistance in weeds.

Anthocyanins are the major pigments responsible for the pomegranate (Punica granatum L.) fruit skin color. The high variability in fruit external color in pomegranate cultivars reflects variations in anthocyanin composition. To identify genes involved in the regulation of anthocyanin biosynthesis pathway in the pomegranate fruit skin we have isolated, expressed and characterized the pomegranate homologue of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1), encoding a WD40-repeat protein. The TTG1 protein is a regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis, and acts by the formation of a transcriptional regulatory complex with two other regulatory proteins: bHLH and MYB. Our results reveal that the pomegranate gene, designated PgWD40, recovered the anthocyanin, PAs, trichome and seed coat mucilage phenotype in Arabidopsis ttg1 mutant. PgWD40 expression and anthocyanin composition in the skin were analyzed during pomegranate fruit development, in two accessions that differ in skin color intensity and timing of appearance. The results indicate high positive correlation between the total cyanidin derivatives quantity (red pigments) and the expression level of PgWD40. Furthermore, strong correlation was found between the steady state levels of PgWD40 transcripts and the transcripts of pomegranate homologues of the structural genes PgDFR and PgLDOX. PgWD40, PgDFR and PgLDOX expression also correlated with the expression of pomegranate homologues of the regulatory genes PgAn1 (bHLH) and PgAn2 (MYB). On the basis of our results we propose that PgWD40 is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development and that expression of PgWD40, PgAn1 and PgAn2 in the pomegranate fruit skin is required to regulate the expression of downstream structural genes involved in the anthocyanin biosynthesis.

Two rapid, nondestructive assays were developed and tested for their potential in differentiating glyphosate-resistant from glyphosate-susceptible biotypes of horseweed. In one assay, leaves of glyphosate-resistant and -susceptible corn, cotton, and soybean plants as well as glyphosate-resistant and -susceptible horseweed plants were dipped in solutions of 0, 300, 600, and 1200 mg ae L−1glyphosate for 3 d and subsequent injury was evaluated. In the second assay, plant sensitivity to glyphosate was evaluated in vivo by incubating excised leaf disc tissue from the same plants used in the first assay in 0.7, 1.3, 2.6, 5.3, 10.6, 21.1, 42.3, and 84.5 mg ae L−1glyphosate solutions for 16 h and measuring shikimate levels with a spectrophotometer. The leaf-dip assay differentiated between glyphosate-resistant and -susceptible crops and horseweed biotypes. The 600 mg L−1rate of glyphosate was more consistent in differentiating resistant and susceptible plants compared with the 300 and 1,200 mg L−1rates. The in vivo assay detected significant differences between susceptible and glyphosate-resistant plants of all species. Shikimate accumulated in a glyphosate dose-dependent manner in leaf discs from susceptible crops, but shikimate did not accumulate in leaf discs from resistant crops and levels were similar to nontreated leaf discs. Shikimate accumulated at high (≥ 21.1 mg ae L−1) concentrations of glyphosate in leaf discs from all horseweed biotypes. Shikimate accumulated at low glyphosate concentrations (≤ 10.6 mg L−1) in leaf discs from susceptible horseweed biotypes but not in resistant biotypes. Both assays were able to differentiate resistant from susceptible biotypes of horseweed and might have utility for screening other weed populations for resistance to glyphosate.

The response of Cuscuta campestris Yuncker, a nonspecific above-ground holoparasite, to amino acid biosynthesis inhibitor (AABI) herbicides, was compared with other resistant and sensitive plants in doseresponse assays carried out in Petri dishes. Cuscuta campestris was found to be much more resistant to all AABI herbicides tested. The I 50 value of C. campestris growth inhibition by glyphosate was eightfold higher than that of transgenic, glyphosate-resistant cotton (RR-cotton). The I 50 value for C. campestris shoot growth inhibition by sulfometuron was above 500 lM, whereas that of sorghum roots was only 0.004 lM. Cuscuta campestris exposed to glyphosate gradually accumulated shikimate, confirming herbicide penetration into the parasite and interaction with an active form of the target enzyme of the herbicide, 5-enolpyruvylshikimate-3-phosphate synthase. More than half of the C. campestris plants associated with transgenic, glyphosate-resistant sugarbeet (RR-sugarbeet) treated with glyphosate or with transgenic, sulfometuron-resistant tomato (SuR-tomato) treated with sulfometuron recovered and resumed regular growth 20-30 days after treatment. New healthy stems developed, followed by normal flowering and seed setting. The results of the current study demonstrate the unique capacity of C. campestris to tolerate high rates of AABI. The mechanism of this phenomenon is yet to be elucidated.