An auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA distribution and synthesis

Abstract

Local concentration gradients of the plant growth regulator auxin (indole-3-acetic acid [IAA]) are thought to instruct the positioning of organ primordia and stem cell niches and to direct cell division, expansion, and differentiation. High-resolution measurements of endogenous IAA concentrations in support of the gradient hypothesis are required to substantiate this hypothesis. Here, we introduce fluorescence-activated cell sorting of green fluorescent protein-marked cell types combined with highly sensitive mass spectrometry methods as a novel means for analyses of IAA distribution and metabolism at cellular resolution. Our results reveal the presence of IAA concentration gradients within the Arabidopsis thaliana root tip with a distinct maximum in the organizing quiescent center of the root apex. We also demonstrate that the root apex provides an important source of IAA and that cells of all types display a high synthesis capacity, suggesting a substantial contribution of local biosynthesis to auxin homeostasis in the root tip. Our results indicate that local biosynthesis and polar transport combine to produce auxin gradients and maxima in the root tip.

Figure 1.

IAA Concentrations in Isolated Protoplasts. (A) IAA concentrations after treatment with or without the transport inhibitors NPA, 1-NOA, NPA + 1-NOA, or NaN₃ during protoplast isolation, normalized to pg IAA/mg protoplasts based on average cell volumes (see Figure 2A). (B) IAA concentrations in isolated protoplasts and in the PBS sorting buffer during simulated sorting for up to 170 min. Each sample consisted of 150,000 isolated protoplasts. Samples were analyzed in triplicate, and error bars indicate sd.

Figure 2.

Protoplast and Cell Diameters of Different Cell Types of the Arabidopsis Root. (A) Average diameters of GFP expressing protoplasts isolated from the different GFP lines used in this study. (B) Cell diameter in relation to protoplast diameter for each of six GFP lines. Diameters of intact cells were calculated from cell volume measurements made on intact roots. Samples were analyzed in triplicate, and error bars indicate sd (n = 10).

Figure 3.

IAA Distribution within the Arabidopsis Root Apex. The data presented in the figure were derived from 14 GFP lines covering all of the different cell types of the root apex. Cell type–specific IAA concentrations were calculated relative to those in the reference population for each GFP cell line, and the color scale indicates the IAA concentration relative to this reference population.

Figure 4.

IAA Distributions in Specific Stele and Root Cap Arabidopsis GFP Lines. (A) Increases in IAA concentrations toward the root apex can be observed in the stele in lines J2501, pWOL:GFP, and Q0990. The extent to which the high IAA level in Q0990 is due to a high IAA concentration in the stele initials or the very high concentration of IAA within its QC cells is unclear. (B) IAA concentration in QC cells, columella initials, columella cells, and in the lateral root cap. The QC cells show a fivefold increase in IAA concentration compared with the reference population, according to both direct measurements on the QC line pAGL42:GFP and measurements on the lines DR5:GFP and M0028 (expressing GFP in the QC), which show four- and threefold higher concentrations compared with their reference populations, respectively. By contrast, IAA concentrations in the J0951 and PET111:GFP lines, which do not contain GFP-expressing QC cells, are similar or lower than those in the reference populations.

Figure 5.

IAA Biosynthesis Rates Are Elevated in the Arabidopsis Root Apex. (A) The GFP-expressing Arabidopsis lines M0028, J2812, pSCR:GFP, and pWOL:GFP were used in this study. The localization of GFP expression is indicated in green for each line. (B) Roots were incubated for 16 h in liquid medium containing 30% ²H₂O and 40 μM NPA, and IAA synthesis rates were measured after protoplast isolation and cell sorting. The IAA biosynthesis rate was 1.5 times higher than the background level in all four lines examined, but none of the four lines showed significantly higher synthesis rate than the others.