Measurement of shikimic acid accumulation in response to glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase is a rapid and accurate assay to quantify glyphosate-induced damage in sensitive plants. Two methods of assaying shikimic acid, a spectrophotometric and a high-performance liquid chromatography (HPLC) method, were compared for their accuracy of recovering known amounts of shikimic acid spiked into plant samples. The HPLC method recovered essentially 100% of shikimic acid as compared with only 73% using the spectrophotometric method. Relative sensitivity to glyphosate was measured in glyphosate-resistant (GR) and non-GR cotton leaves, fruiting branches, and squares (floral buds) by assaying shikimic acid. Accumulation of shikimic acid was not observed in any tissue, either GR or non-GR, at rates of 5 mM glyphosate or less applied to leaves. All tissues of non-GR plants accumulated shikimic acid in response to glyphosate treatment; however, only fruiting branches and squares of GR plants accumulated a slight amount of shikimic acid. In non-GR cotton, fruiting branches and squares accumulated 18 and 11 times, respectively, more shikimic acid per micromolar of translocated glyphosate than leaf tissue, suggesting increased sensitivity to glyphosate of reproductive tissue over vegetative tissue. GR cotton leaves treated with 80 mM of glyphosate accumulated 57 times less shikimic acid per micromolar of translocated glyphosate than non-GR cotton but only 12.4- and 4-fold less in fruiting branches and squares, respectively. The increased sensitivity of reproductive structures to glyphosate inhibition may be due to a higher demand for shikimate pathway products and may provide an explanation for reports of fruit abortion from glyphosate-treated GR cotton.

Glyphosate treatments to glyphosate-resistant (GR) cotton have been associated with poor pollination and increased boll abortion. Anatomical studies were conducted to characterize the effect of glyphosate treatments on the development of male and female reproductive organs of cotton flowers at anthesis. In comparison with nontreated plants, glyphosate applied at both the four-leaf stage postemergence (POST) and at the eight-leaf stage POST directed inhibited the elongation of the staminal column and filament, which increased the distance from the anthers to the receptive stigma tip by 4.9 to 5.7 mm during the first week of flowering. The increased distance from the anthers to the stigma resulted in 42% less pollen deposited on stigmas of glyphosate-treated plants than in nontreated plants. Moreover, pollen from glyphosate-treated plants showed numerous morphological abnormalities. Transmission electron microscopy showed the presence of large vacuoles, numerous starch grains, and less organized pockets of the endoplasmic reticulum containing fewer ribosomes in pollen from glyphosate-treated plants than from nontreated plants. Pollen development in glyphosate-treated plants is likely inhibited or aborted at the vacuolate microspore and vacuolate microgamete stages of microgametogenesis, resulting in immature pollen at anthesis. Although stigmas from glyphosate-treated plants were 1.2 to 1.4 mm longer than those from nontreated plants, no other anatomical differences in stigmas were visibly evident. The presence of the GR 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) enzyme from Agrobacterium sp. strain CP4 was quantified in reproductive and vegetative tissues using enzyme-linked immunosorbent assay. The content of CP4-EPSPS in the stigma, anther, preanthesis floral bud (square), and flower petals was significantly less than that in the vegetative leaf tissue. Glyphosate effects on the male reproductive development resulting in poor pollen deposition on the stigma, as well as production of aborted pollen with reduced viability, provide a likely explanation for reports of increased boll abortion and pollination problems in glyphosate-treated GR cotton.

The influence of herbicide placement and plant growth stage on the absorption and translocation patterns of 14C-glyphosate in glyphosate-resistant cotton was investigated. Plants at four growth stages were treated with 14C-glyphosate on a 5-cm2 section of the stem, which simulated a postemergence-directed spray (PDS) application, or on the newest mature leaf, which simulated a postemergence (POST) application. Plants were harvested 3 and 7 d after treatment and divided into the treated leaf or treated stem, mature leaves, immature leaves and buds, stems, roots, fruiting branches (including the foliage on the fruiting branch), squares, and bolls. The PDS versus POST application main effect on absorption was significant. Absorption of 14C-glyphosate applied to stem tissue was higher in PDS applications than in POST applications. Plants receiving PDS applications absorbed 35% of applied 14C-glyphosate, whereas those receiving POST applications absorbed 26%, averaged over growth stages at application. Absorption increased from the four-leaf growth stage to the eight-leaf stage in POST applications but reached a plateau at the eight-leaf stage. Plants with PDS applications showed an increase in absorption from the four- to eight- to twelve-leaf stages and reached a plateau at the 12-leaf stage. Translocation of 14C-glyphosate to roots was greater at all growth stages with PDS treatments than with POST treatments. Herbicide placement did not affect translocation of 14C-glyphosate to squares and bolls. Squares and bolls retained 0.2 to 3.7% of applied 14C-glyphosate, depending on growth stage. Separate studies were conducted to investigate the fate of foliar-applied 14C-glyphosate at the four- or eight-leaf growth stages when harvested at 8- or 10-leaf, 12-leaf, midbloom (8 to 10 nodes above white bloom), and cutout (five nodes above white bloom, physiological maturity) stages. Thirty to 37% of applied 14C-glyphosate remained in the plant at cutout in four- and eight-leaf treatment stages, respectively. The concentration of 14C-glyphosate in tissue (Bq g−1 dry weight basis) was greatest in mature leaves and immature leaves and buds in plants treated at the four-leaf stage. Plants treated at the eight-leaf stage and harvested at all growth stages except cutout showed a higher concentration of 14C-glyphosate in squares than in other plant tissue. Accumulation of 14C-glyphosate in squares reached a maximum of 43 Bq g−1 dry weight at harvest at the 12-leaf stage. This concentration corresponds to 5.7 times greater accumulation of 14C-glyphosate in squares than in roots, which may also be metabolic sinks. These data suggest that reproductive tissues such as bolls and squares can accumulate 14C-glyphosate at higher concentrations than other tissues, especially when the herbicide treatment is applied either POST or PDS during reproductive stages (eight-leaf stage and beyond).