Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture

Abstract

Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with 13C6-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR,) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2-phenylindole stained cells showed that jsR, has the same amount of DNA per nucleus as the parent line (PL). All jsR, cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR, at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR, had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR, and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR, had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity. sist of cloned vegetative plants. Our cultures of Lemna gibba G-3 increase in frond2 number and fresh weight at a logrithmic rate throughout a 45 d culture cycle. In this report we show that, during the culture cycle, endogenous IAA levels vary from only a few ng/g FW3 to several hundred ng/g FW.Size variation in selected varieties of higher plants has been attributed to a variety of genetic determinants such as ploidy level (3), root physiology (20), and hormonal variations (20,22 injector was at 260°C and the oven was programmed with a 2 min isothermal hold at 140°C then increased at 5°C/min. Ultrapure He, after passage through an oxygen trap and through a molecular sieve (Linde 4A), was used as carrier gas at a flow rate of 1 ml/min.Ester and amide conjugates were determined by using selective base hydrolysis as previously described (12). Data presented are the average of four replicate experiments. RESULTSGrowth Characteristics. L. gibba G-3 is an aquatic angio §perm that grows well in sterile culture on minimal media. Under short day conditions propagation is entirely vegetative. Using E media, which consists of salts and contains 1% sucrose, flasks planted with Lemna support plant growth at a logrithmic rate for over 45 d, with no apparent lag phase or plateau (Fig. 2). Growth rate does not account for the difference in size of PL and jsR,.Lemna fronds exhibit a determinant growth characteristic. Each frond will produce approximately 15 daughter fronds and then the mother frond senesces. In our cultu...

Selection and Characterization of [alpha]-Methyltryptophan-Resistant Lines of Lemna gibba Showing a Rapid Rate of Indole-3-Acetic Acid Turnover

“…HPLC was carried out on a C,, 50 mm X 4.6 mm Phenomenex Ultracarb 30 5p ODS column The sample was eluted isocratically using a mobile phase of 25% methanol containing 1% (v/v) acetic acid. The IAA fraction was methylated using diazomethane and analyzed by GC-MS selected ion monitoring as described by Slovin and Cohen (1988).…”

Indole-3-Acetic Acid Metabolism in Lemna gibbaUndergoes Dynamic Changes in Response to Growth Temperature

“…IAA levels can vary greatly over the culture cycle even though the growth rate in L. gibba remains constant (Slovin and Cohen, 1988). These observations may be related to changing culture conditions such as population density.…”

“…Therefore, a major goal in studies of environmental physiology is to understand the link between environment and phytohormone signaling. Advances in methodology (Rapparini et al, 1999) as well as an improved understanding of auxin biochemistry have enabled us to use the L. gibba model system (Slovin and Cohen, 1988;Tam et al, 1998) for detailed analysis of the interaction of environment and auxin metabolism. The results reported in this paper show that the temperature of the growth environment alters two aspects of IAA metabolism, turnover and biosynthetic route.…”

“…For example, for the 5°C treatment plants were grown at 25°C for 14 d and were then transferred to 5°C for 10 d. For the 30°C treatment, plants were grown at 25°C for 6 d, and were then transferred to 30°C for 10 d. However, plants were grown for 16 d at 25°C and were transferred to 35°C for only 3 d because we had determined that the plants stop growing after about 4 d at 35°C. These protocols were necessary because previous studies had shown that IAA levels were not constant throughout the culture period at 25°C (Slovin and Cohen, 1988).…”

“…Cultures were kept under continuous light that was provided by a mixture of cool-white fluorescent and incandescent lamps at 25 Em Ϫ2 s Ϫ1 (Slovin and Cohen, 1988).…”