Involvement of auxin and brassinosteroid in the regulation of petiole elongation under the shade

Abstract

Plants grown under a canopy recognize changes in light quality and modify their growth patterns; this modification is known as shade avoidance syndrome. In leaves, leaf blade expansion is suppressed, whereas petiole elongation is promoted under the shade. However, the mechanisms that control these responses are largely unclear. Here, we demonstrate that both auxin and brassinosteroid (BR) are required for the normal leaf responses to shade in Arabidopsis (Arabidopsis thaliana). The microarray analysis of leaf blades and petioles treated with end-of-day far-red light (EODFR) revealed that almost half of the genes induced by the treatment in both parts were previously identified as auxin-responsive genes. Likewise, BR-responsive genes were overrepresented in the EODFR-induced genes. Hence, the auxin and BR responses were elevated by EODFR treatment in both leaf blades and petioles, although opposing growth responses were observed in these two parts. The analysis of the auxin-deficient doc1/big mutant and the BR-deficient rot3/cyp90c1 mutant further indicates that auxin and BR were equally required for the normal petiole elongation response to the shade stimulus. In addition, the spotlight irradiation experiment revealed that phytochrome in leaf blades but not that in petioles regulated petiole elongation, which was probably mediated through regulation of the auxin/BR responses in petioles. On the basis of these findings, we conclude that auxin and BR cooperatively promote petiole elongation in response to the shade stimulus under the control of phytochrome in the leaf blade.

Figure 1.

Effects of the light treatments on the growth of leaf blades and petioles. Leaf blade area (left) and petiole length (right) were determined in the wild-type seedlings grown under continuous white light (cWL; 70 μmol m⁻² s⁻¹) or short days without (−) or with (+) EODFR (50 μmol m⁻² s⁻¹ for 5 min). The short days consisted of 10-h-white-light (100 μmol m⁻² s⁻¹)/14-h-dark cycles. Fourth leaves were used for the size measurement (n ≥ 15, mean ± sd). Asterisks indicate significant differences from the white light controls (* P < 0.05, Student's t test).

Figure 2.

Microarray analysis of differential gene expression across three light conditions. A, The light regimes. An FR pulse (50 μmol m⁻² s⁻¹ for 5 min) was given at the end of the day. cWL, Continuous white light. B, Flow chart showing the number of genes that were statistically and robustly induced or repressed in response to the light treatments.

Figure 3.

Comparative analysis of EODFR- and dark-induced genes in leaf blades and petioles. A, Venn diagrams illustrating the different responses to the EODFR and dark treatments. B, Venn diagrams illustrating the different responses in leaf blades and petioles. The numbers of genes belonging to each category are shown.

Figure 4.

The percentages of auxin- and BR-responsive genes found in the EODFR-induced (A) and dark-induced (B) genes. Pie charts show the numbers and percentages (in parentheses) of genes that are induced only by auxin (blue), only by BR (red), and by both auxin and BR (white). Auxin- and BR-responsive genes were defined according to Nemhauser et al. (2006).

Figure 5.

The growth response to EODFR in the phyB, doc1, and rot3 single and multiple mutants. The petiole lengths (A) and areas of the leaf blade (B) of the matured fourth rosette leaves were determined in the wild type (WT), phyB, doc1, phyBdoc1, rot3, phyBrot3, doc1rot3, and phyBdoc1rot3. These seedlings were grown under short days without (−) or with (+) EODFR as described in Figure 1. Fourth leaves were used for the size measurement (n ≥ 15, mean ± sd). Asterisks indicate significant differences from the respective EODFR controls (* P < 0.05, Student's t test).

Figure 6.

Up-regulation of the auxin- and BR-responsive genes in response to EODFR. The mRNA levels were determined in the leaf blade and petiole of the wild-type (WT), doc1, and rot3 plants treated with (+) or without (−) EODFR as described in Figure 2A. A, Auxin-responsive genes: GH3.3, IAA6, and SAUR68. B, Auxin- and BR-responsive genes: IAA19, XTH19, and XTH22. C, BR-responsive genes: At5g24580, F8H, and At3g28200. The transcription levels were quantified by real-time RT-PCR and normalized to UBQ10 (see “Materials and Methods”). Data are expressed in relative units and represented as means ± sd (n = 3).

Figure 7.

Effect of FR spotlight irradiation on the leaf blades and petioles. Fifth rosette leaves of 14-d-old plants grown under continuous white light were treated without (−) or with a FR pulse on the whole plants (w), leaf blade (b), petiole (p), or leaf blade plus petiole (b+p). A, Increases in the petiole (black bars) and blade (gray bars) lengths. The increments were determined after 2-h rounds of the EODFR treatment. Data are represented as means ± sd (n ≥ 15). Asterisks indicate significant differences from the respective EODFR controls (* P < 0.05, Student's t test). B, GH3.3 and IAA6 mRNA levels in the leaf blade and petiole in response to a FR pulse given as described in A. The mRNA levels were quantified by real-time RT-PCR and normalized to UBQ10 (see “Materials and Methods”). Data are represented as means ± sd (n =3).

Figure 8.

Effect of NPA on petiole elongation under different light conditions. A, The leaves were excised and grown on agar plates containing NPA at various concentrations for 48 h under high (R [10 μmol m⁻² s⁻¹]) or low R:FR (R [10 μmol m⁻² s⁻¹], FR [90 μmol m⁻² s⁻¹]) conditions. B, The wild-type seedlings were sprayed with the NPA solution and then grown for 48 h as in A. The fifth rosette leaves were used (n ≥ 15, mean ± sd). Asterisks indicate significant differences from the mock controls (* P < 0.05, Student's t test).