Abstract
The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling.
Highlights
The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers
We found that portion of [13C6]IAA taken up by Arabidopsis hypocotyls moved basipetally and that the cm[1o3anCjvo6]erIirAttyeAdo.ftIonth[ce1o3tCnra1t]rnIaAssptA,oaratlenaddrg[ee1s3Ctpe6or]-rIltAiinoAknerodefm[[113a3CiCn11]e]IdIBBAaAsdwfruearesing the transport period, and the proportion of the laopwpeliredtha[1n3Cth1]eIBpArotproarntsiopnorotefdapbpalsiiepdet[a13lCly6]wIAaAs much transported basipetally, suggesting that long-distance transport of IBA has a much smaller effect on IBA pools than transport of IAA has on IAA pools
Five plants were needed for facile detection otddIAiAfooestAnn[se1ohc3oCtrotrfew6aad[]c1gIn3esAaCerirn,A1p]abaIF,BnlriwogAadcuthekrtailhe,lnyee2ud; Bt2n[h[,02l1Huaw3Cpbs4h,el]1ae]ItlIAhennAdetA[sA1[3e1Cwi3ndnC6dee]t6Irero]AeirIgvAnAeneanAdewloefstadurrtsoeasamadcnIdedArfd[ao1Acer3roCdd,ud1[i]wlne1Id3BtCetehbArc6eee-]. distinguished from endogenous IAA, and the amount foIrAfoAm[13Cin[61t]3eICAr6nA]aIAl cAostualwnddaabsredaq.lsuPoaontmteinfiotenidaitlobr[ae1s3dCed6]bIouBntAtwhdeaesr[i2vHneo4d]t detected, which may be due to a low rate of conversion Soifm[1i3lCar6]lyIB, wAhthenat[m13Cay1]IrBeqAuwiraesmaodrdeetdisisnuethfeorddoentoerctaigoanr. block (Fig. 2C), both IAA and IBA were monitored, and their retention times on GC were about 1 min apart
Summary
The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments establish a method for quantifying the movement of small molecules in plants using stable isotope labeling. Indole-3-butyric acid (IBA), which has had a long history of use as a synthetic auxin to promote root initiation, has been identified as an endogenous compound in a variety of plants (for review, see Ludwig-Muller, 2000), including Arabidopsis
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