Role for apyrases in polar auxin transport in Arabidopsis

Abstract

Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis (Arabidopsis thaliana). Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ectonucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [(3)H]indole-3-acetic acid in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues, polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNA interference. In the hypocotyl assays, the basal halves of APY-suppressed hypocotyls contained considerably lower free indole-3-acetic acid levels when compared with wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a green fluorescent protein fluorescence signal encoded by a DR5:green fluorescent protein construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together, these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport.

Figure 1.

Polar auxin transport is inhibited in Arabidopsis hypocotyls after the induction of apyrase RNAi by estradiol and promoted in hypocotyls from seedlings overexpressing APY1 and APY2. A, Estradiol-treated RNAi R2-4A seedlings grown for 6 d in the dark and then transferred to light for 2 d show inhibited growth compared with Ws, noninduced RNAi R2-4A, and estradiol-treated Ws seedlings. B, Auxin transport is similar in noninduced genotypes, and there is no difference in the ability of NPA to inhibit basipetal auxin transport among noninduced genotypes. C, Basipetal auxin transport is greatly reduced after suppression of APY2 by the induction of mRNA interference with estradiol treatment. D, Basipetal auxin transport is promoted in APY1-OE and APY2-OE hypocotyls but is unaffected in the apy1 and apy2 single knockout (KO) lines. NPA inhibits basipetal auxin transport in hypocotyls of all genotypes but to a lesser degree in hypocotyls of the estradiol-treated RNAi R2-4A line. se values are marked by vertical bars. In B, n = 10 for all groups; in C and D, n = 10 for basipetal groups and n = 5 for acropetal and NPA groups. These results are representative of three or more biological repeats. Statistically evaluated differences between samples are indicated by asterisks (Student’s t test; **P < 0.05, *P < 0.07). [See online article for color version of this figure.]

Figure 2.

Free IAA levels are increased in the shoot apices and decreased in the basal halves of hypocotyls of Arabidopsis seedlings after the induction of apyrase RNAi by estradiol. Free IAA levels were significantly decreased in the basal half of R2-4A (+estradiol) hypocotyls compared with the basal halves of R2-4A (−estradiol) or Ws wild-type (WT; +/−estradiol) hypocotyls, and the increase of free IAA found in the apices of R2-4A (+estradiol) hypocotyls was only marginally insignificant. Free IAA in the apical half of hypocotyls showed no difference among the groups analyzed. A shows a graphical representation of the data shown in B. se values are marked by vertical bars. These results are representative of three or more biological repeats. Statistically evaluated differences between samples are indicated by asterisks (Student’s t test; **P < 0.002, *P < 0.07; n = 3). FW, Fresh weight.

Figure 3.

Polar auxin transport is inhibited in primary roots of Arabidopsis after the induction of apyrase RNAi by estradiol. IAA transport was measured in both the basipetal (shootward) and acropetal (rootward) directions in 8-d-old seedlings 4 d after transfer to estradiol. Basipetal IAA transport was reduced in R2-4A roots when induced by estradiol. The average and se values are reported for greater than 42 seedlings in the basipetal assay and greater than 60 seedlings in the acropetal assay and are a summary of three biological repeats. Basipetal IAA transport levels in the wild type averaged 4.2 fmol, and acropetal IAA transport values in the wild type averaged 8.8 fmol. A statistically significant difference between the wild type and the RNAi line is indicated by the asterisk (Student’s t test; *P < 0.05).

Figure 4.

Suppression of APY1 in the apy2 mutant alters the morphology of roots and the expression of the auxin response reporter DR5:GFP. Shown are primary roots of Arabidopsis seedlings grown for 6 d in the light. A, C, E, and G, Roots of DR5:GFP R2-4A treated for 3 d (A and C) or 4 d (E and G) with the estradiol inducer. B, D, F, and H, DR5:GFP Ws roots treated with estradiol for 3 d (B and D) or 4 d (F and H). These results are representative of 10 or more biological repeats. Bars = 100 µm. WT, Wild type.

Figure 5.

Scanning electron microscopy images of 6-d-old estradiol-treated Ws wild-type and RNAi R2-4A mutant seedlings showing root swelling and ectopic root hair development. A, Enlarged apical zone of Ws wild-type root. B, Enlarged apical zone of R2-4A root. These results are representative of three or more biological repeats. Bars = 200 µm.

Figure 6.

A and B, Fluorescent GFP signals in horizontally positioned primary roots of wild-type (WT; A) and R2-4A (B) plants expressing DR5:GFP. Both wild-type and R2-4A plants were treated with estradiol, which induced the suppression of apyrase expression by RNAi in the R2-4A mutant. C to F, GFP signals in horizontally positioned primary roots of wild-type plants expressing DR5:GFP before and after treatment with 800 µm ATPγS (C and D) or 7.5 µg mL⁻¹ NGXT1913 (E and F). Arrows indicates the lower flank of the primary root, where the GFP signal is evident in the epidermal cells of the wild-type roots but not in the R2-4A roots and not in treated roots. These results are representative of 10 or more biological repeats. Signals were assayed 5 h after roots moved to the horizontal position. Bars = 50 µm.