Simultaneous Gas Chromatography-Mass Spectrometry Quantification of Endogenous [¹²C]- and Applied [¹³C]Indole-3yl-Acetic Acid Levels in Growing Maize Roots

Abstract

(2,6,24,30). Research has so far been oriented into three main directions: (a) IAA labeled with radioactive isotopes (3H, 14C) was applied to plant tissue and the pattern of IAA metabolism analyzed (11, 17); (b) enzymes, which oxidize IAA (15), hydrolyze IAA conjugates (4, 10) and bind IAA to conjugates (12) were also partially characterized; (c) the products of these in vitro enzymatic reactions and also some of the endogenous indolic compounds were isolated and identified (16,23,28). Moreover, the level of IAA in a particular time and part of the root reflects the steady-state balance (9), on the one hand, between the rates of anabolism (including hydrolyses of IAA conjugates) (6,24) and the rates of catabolism (oxidation and conjugation) (18,28) and, on the other hand, ofthe rates and directions oftransport fluxes (acropetal and basipetal transport) (13, 19) of IAA.Few workers, however, have been concerned with the correlation between IAA metabolism and plant growth (3) and none (to our knowledge) have tried to follow the behavior of both endogenous and applied IAA after an IAA treatment. In this current work, a stable isotope of IAA (labeled with 6 atoms of 13C) was therefore employed not as the more usual internal standard (7) but for hormone treatments of growing maize roots. The aim of the present investigation was to determine simultaneously, using GC-SIM2-MS, the levels of endogenous ['2C]IAA and applied ['3C]IAA in three different zones (apical, elongating, differentiating) and thus to correlate the amount of the two forms of IAA to root growth.Several reports have established, using GC-MS, the natural occurrence of IAA and its role in the regulation of root growth (5,8,22,25,29). Two main approaches to this problem have generally been used. First, different techniques of IAA application have established that IAA inhibits the growth ofnormal roots (1) but might stimulate growth of auxin-depleted roots (20). Both effects depend on the amount of IAA (22) and the localization of the treatment along the root (14). Second, quantifications of levels of endogenous IAA in maize roots have shown an inverse relation (modified during germination) between the growth rate and the level of free and ester-bound IAA in the elongation zone (22,29).

Quantification of indole-3-acetic acid in untransformed and Agrobacterium rhizogenes-transformed pea roots using gas chromatography mass spectrometry

“…The extraction of free IAA was done according to Saugy and Pilet (1987) and Meuwly and Pilet (1991), with some minor modifications. To the dried plant material were added 20 ng [2Hs]IAA as internal standard and 3 ml of ice-cold sodium acetate-acetic acid buffer (10 mM, pH 4.8).…”

“…Gas-chromatography conditions have been previously described (Meuwly and Pilet 1991). The values of m/z of the base peaks were monitored (326 for IAA-Me-HFB and 331 for 2Hs-IAA--Me-HFB ).…”

Assimilation of exogenous 2′-14C-indole-3-acetic acid and 3′-14C-tryptophan exposed to the roots of three wheat varieties

“…They can be assimilated by the roots (intact or excised) (Meuwly and Pilet, 1991;Thurman and Street, 1962), utilized as a carbon source by soil microorganisms (Arshad and Frankenberger, 1990), or metabolized by root tissues (Beffa et al, 1990;Meuwly and Pilet, 1991). They can be assimilated by the roots (intact or excised) (Meuwly and Pilet, 1991;Thurman and Street, 1962), utilized as a carbon source by soil microorganisms (Arshad and Frankenberger, 1990), or metabolized by root tissues (Beffa et al, 1990;Meuwly and Pilet, 1991).…”

Local treatment with indole-3-acetic acid induces differential growth responses in Zea mays L. roots

“…(i) Some mechanical properties of the cell walls (Masuda and Pilet 1983;Taiz 1984;Hofer and Pilet 1986) or the small size of cells (Darbelley and Perbal 1984;BalOska et al 1990) might prevent any compression inherent in a curvature response (Barlow and Rathfelder 1985). (ii) The disappearance of the active free form of IAA through the intensive (Nonhebel et al 1985) and rapid (Meuwly and Pilet 1991) process of metabolism described in maize roots might reduce drastically the source of the growth signal in this region. (iii) There may be a reduced availability of IAA receptors (Rubery 1981 ;Venis 1985;Jones 1990) within the cells of this acropetal region of the elongating zone.…”

“…No characterization of the metabolites of IAA was attempted in these experiments, although applied IAA is known to metabolize rapidly in maize roots immersed in radioactive IAA (Nonhebel et al 1985) and, to a lesser extent, after application of IAA-loaded beads (Pilet and Meuwly 1986). It would be difficult to evaluate the total amount of free IAA (endogenous and applied) present in the root tissue at the end of an IAA treatment as the rate of metabolism of each type (applied and endogenous) might differ significantly (Meuwly and Pilet 1991). The uptake of IAA (expressed in pg/root from the radioactive values) therefore represents the total amount of IAA, not just the free IAA, which has entered into the root during the 4 h treatment.…”