APYRASE1/2 mediate red light-induced de-etiolation growth in Arabidopsis seedlings

Abstract

In etiolated seedlings, red light (R) activates phytochrome and initiates signals that generate major changes at molecular and physiological levels. These changes include inhibition of hypocotyl growth and promotion of the growth of primary roots, apical hooks, and cotyledons. An earlier report showed that the sharp decrease in hypocotyl growth rapidly induced by R was accompanied by an equally rapid decrease in the transcript and protein levels of two closely related apyrases (APYs; nucleoside triphosphate-diphosphohydrolases) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, enzymes whose expression alters auxin transport and growth in seedlings. Here, we report that single knockouts of either APY inhibit R-induced promotion of the growth of primary roots, apical hooks, and cotyledons, and RNAi-induced suppression of APY1 expression in the background of apy2 inhibits R-induced apical hook opening. When R-irradiated primary roots and apical hook-cotyledons began to show a gradual increase in their growth relative to dark controls, they concurrently showed increased levels of APY protein, but in hook-cotyledon tissue, this occurred without parallel increases in their transcripts. In wild-type seedlings whose root growth is suppressed by the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the R-induced increased APY expression in roots was also inhibited. In unirradiated plants, the constitutive expression of APY2 promoted both hook opening and changes in the transcript abundance of Small Auxin Upregulated RNA (SAUR), SAUR17 and SAUR50 that help mediate de-etiolation. These results provide evidence that the expression of APY1/APY2 is regulated by R and that APY1/APY2 participate in the signaling pathway by which phytochrome induces differential growth changes in different tissues of etiolated seedlings.

Figure 1

Suppression of APY expression impairs R-induced hook opening, cotyledon expansion, and root growth. A, Apical hook opening of 3.5-day-old dark-grown seedlings, 2, 4, 6, 8, and 12 h after R treatment of WT Ws, (−E) R2-4A mutants without estradiol, and (+E) R2-4A mutants with estradiol. B, Increase in the growth of cotyledons of 2.5-day-old, etiolated seedlings induced by continuous R (30 mol m⁻² s⁻¹) is significantly inhibited in apy1 and apy2. Cotyledon area was measured at 24 h time intervals. C, Cotyledon area measurements with estradiol inducer added to R2-4A mutants at the beginning of the 3.5 days dark period and imaged 1, 2, and 3 days after R, (−E) R2-4A mutants without estradiol, and (+E) R2-4A mutants with estradiol. D, Root length measured at 24-h time intervals. Estradiol inducer was added to R2-4A mutants to suppress APY1 expression by RNAi after the 2.5 days dark period. E, Estradiol inducer is added to R2-4A mutants (+E) at the beginning of the 3.5 days dark period, maximal suppression of APY1 is evident when R treatment begins at the end of the dark period, and R does not induce any increase in primary root growth thereafter. For all panels (A–E), shown is a representative result that was observed in samples independently grown on three separate plates, each of which had an n value of at least 7, and different letters above the bars indicate values that differ significantly from each other (P < 0.05, Student’s t test, error bars are S.E.).

Figure 2

R induces changes in the level of APY protein in primary roots. R induces a decrease in APY protein expression in primary roots at early time points (A), and an increase 24 h after R exposure (B). After seedlings were grown for 2.5 days in darkness, they were irradiated with continuous R for 6, and 12 h (A), or maintained in darkness or irradiated with continuous R for 24 h (B). APY1 and APY2 levels were detected by anti-APY antibody, and the same blot was stripped and re-probed with anti-HIS3 antibody for equal loading control. Densitometry measurements of APY and HIS3 levels were measured in cotyledons (A), or in roots (B) by ImageJ.

Figure 3

R induces changes in the abundance of APY transcripts in roots and hook-cotyledon tissues. R induces changes in the transcript levels of APY1 (A) and APY2 (B) in hook-cotyledon and root tissues of dark-grown Ws WT seedlings after 24 and 72 h of continuous light treatment. Respective dark samples at 24 and 72 h time points are used as calibrators. Experiment performed with three biological repeats, each with three technical replicates. Error bars represent the se. Student’s t test was applied. * Denotes significantly different expression from calibrator (dark) at P < 0.05.

Figure 4

R induces an increase in the level of APY protein in apical hook-cotyledon tissue. The increase in the level of APY protein induced by R in apical hook-cotyledons is gradual, rising to consistently 1.3-fold or higher at and after 6 h of R irradiation. After Ws WT seedlings were grown for 2.5 days in darkness, they were irradiated with continuous R for 2, 4, 6,12, 24, and 72 h. APY1 and APY2 levels were detected by anti-APY antibody, and the same blot was stripped and re-probed with anti-HIS3 antibody for equal loading control. Densitometry measurements of APY and HIS3 levels were measured by ImageJ.

Figure 5

On media without sucrose added, R does not induce a significant increase in APY protein levels if seedling photosynthesis is inhibited by DCMU, and R significantly promotes root growth A, Ws WT seedlings grown in dark, or transferred to continuous R after 2.5 days growth in dark, were grown under R with no treatment, with DMSO (0.001%V/V) or with 50 μM DCMU dissolved in DMSO (0.001% V/V) for 72 h. APYl and APY2 levels were detected by anti-APY antibody, and the same blot was stripped and reprobed with anti-HIS3 antibody for equal loading control. Densitometry measurements of APY and HIS3 levels were measured by ImageJ, and the signal ratio is expressed relative to the dark sample. B, The length of roots in both R-treated and dark-grown apy1 and apy2 seedlings is significantly less than that observed in Ws WT seedlings. Shown is a representative result that was observed in samples independently grown on three separate plates, each of which had an n value of 10. The R treatment in both (A) and (B) was for 24 h. Different letters above the bars indicate values that differ significantly from each other (P < 0.05, Student’s t test; error bars are se).

Figure 6

Constitutive expression of APY1 or APY2 promote hook opening in etiolated seedlings. A, 2.5-day-old dark-grown APY1 OE and APY2 OE seedlings without sucrose in the growth medium show significant apical hook opening without the R light stimulus. Shown is a representative result that was observed in samples independently grown on three separate plates, each of which had an n value of 11. The R treatment in both (A) and (B) was for 24 h. Different letters above the bars indicate values that differ significantly from each other (P < 0.05, Student’s t test; error bars are se). B, Representative WT Ws, APY1 OE and APY2 OE seedlings. Bar 5 mm. C, Illustration of an A. thaliana apical hook measurement from the mid-line to the tip of the cotyledon.

Figure 7

Constitutive expression of APY2 promotes light-induced changes in SAUR50 and SAUR17 gene expression in the hook-cotyledon tissue of 4-day-old, etiolated seedlings. Relative expression levels of SAUR50 and SAUR17 (A) WT Ws in dark versus after 3 h of white light. B, WT Ws in dark versus APY2 OE in dark. The data represent average of 4–10 technical replicates of RNA extracted from tissue pooled from five independently grown plates of seedlings. * Denotes significantly different expression from the WT Ws expression level calibrator (P < 0.05, Student’s t test, error bars are se).