SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation

Abstract

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.

Figure 1.

Effects of Pi availability and the application of 0.05 μm IAA on the root system architecture of wild-type (WT) and siz1 seedlings. The seedlings grown on basal medium for 3.5 d were transferred onto medium containing high Pi (1.25 mm KH₂PO₄; +Pi) or low Pi (0.0125 mm KH₂PO₄; −Pi), and the growth of PR (A) and density of LR (B) were scored each day. The LR density was calculated by visible LR (more than 1 mm length) divided by the PR length. To monitor the effect of IAA on the root system architecture under high or low Pi availability, the seedlings were transferred onto medium containing 0.05 μm IAA (C and D). The growth of PR after the transfer and the density of the LR were scored. All values shown represent averages of 12 seedlings ± se.

Figure 2.

Application of IAA reduces PR growth and increases LR density, whereas NPA treatment decreases LR density in wild-type (WT) and siz1 seedlings. Seedlings were grown on basal medium containing Pi for 3.5 d before being transferred onto medium containing high Pi (1.25 mm KH₂PO₄; +Pi) or low Pi (0.0125 mm KH₂PO₄; −Pi) and grown for 7 d. PR growth and LR density of wild-type and siz1 seedlings that were grown on various concentrations of IAA (A and B) or NPA (C and D) were scored. All values represent means of 12 or more seedlings ± se.

Figure 3.

Seedlings overexpressing YUCCA1 show a hyperresponse phenotype to Pi deficiency in the root system architecture. Wild-type (WT) and siz1-2 seedlings, as well as seedlings overexpressing YUCCA1 and YUCCA6, were incubated on Pi-sufficient (A) or Pi-deficient (B) medium. The flavin monooxygenases gene, YUCCA, plays an important role in auxin biosynthesis (Zhao et al., 2001; Cheng et al., 2006; Kim et al., 2007). Seedlings overexpressing YUCCA1, but not seedlings overexpressing YUCCA6, exhibited a shorter PR and longer LR. The experimental procedure is the same as that described in Figure 1. Bars = 10 mm. Quantitative data of PR growth and LR density are shown in Supplemental Figure S2. [See online article for color version of this figure.]

Figure 4.

DR5::uidA expression in PR tips and LR tips. A, No significant change was observed in high-Pi conditions. B, DR5::uidA expression in the PR tips of the wild type (WT) was increased after 3 d of growth under the low-Pi condition and was then decreased. In contrast, the expression in siz1 was increased 1 d after transfer onto Pi-deficient medium. C and D, DR5::uidA expression in the LR tips of wild-type and siz1-2 seedlings, respectively, after 1, 2, 3, and 5 d of treatment with Pi starvation. Three representative LR tips are shown. E and F, DR5-GUS accumulation in the LR tips of wild-type and siz1-2 seedlings, respectively, was observed at 7, 9, 11, and 14 d after the transfer to low-Pi medium. The top panels are two representative younger LR tips, and the bottom panels are older LR tips. Bars = 100 μm. [See online article for color version of this figure.]

Figure 5.

Recovery of PR. A, Nine days after the Pi starvation treatment, the seedlings were transferred onto the medium that was supplemented with 1.25 mm KH₂PO₄ and incubated for 5 d. The blue and red arrowheads indicate the start (just after transfer onto Pi-supplied medium) and end (5 d after the transfer) points, respectively. The root growth of the LR was observed in both wild-type (WT) and siz1-2 seedlings. B and C, Nine days after the treatment of the Pi-deficient (−Pi) condition, the PR of wild-type and siz1-2 seedlings was stained with propidium iodide, as described previously (Miura et al., 2010). B, In wild-type seedlings, only the cell membranes were stained because intact cell membranes block the penetration of propidium iodide. C, In siz1-2 seedlings, the cells were disordered, and propidium iodide can pass through damaged cell membranes and intercalate with DNA. The arrowheads indicate nuclei intercalated with the propidium iodide. Bars = 10 mm (A) and 100 μm (B and C). [See online article for color version of this figure.]

Figure 6.

A, pCycB1;1::uidA expression in PR tips 7 d after the transfer into high- and low-Pi conditions. The pCycB1;1::uidA expression in the PR tips of siz1-2 seedlings grown under Pi starvation was substantially decreased. B, pSIZ1::uidA expression in PR tips at 3 and 7 d after the transfer onto Pi-sufficient and starvation media, respectively. The expression of pSIZ1::uidA at 3 d after the transfer was substantially decreased. WT, Wild type. Bars = 100 μm. [See online article for color version of this figure.]

Figure 7.

Functional categorization of SIZ1-regulated genes. A, Cluster analysis of transcripts, which were up- or down-regulated in siz1-2, before and after Pi starvation, with NAA, IAA, and Pi-deficient treatments. B and C, The expression data after NAA, IAA, and Pi-deficient treatments were obtained from Genevestigator. Two of the clusters were magnified. The genes, which were up-regulated in siz1-2 and under Pi deficiency but were not significantly altered by auxin, were included in the cluster (B). In another cluster, the genes were up-regulated in siz1-2 and by Pi starvation and auxin (C). The genes encoding glycosyl hydrolase and glycosyl transferase were included in this cluster (C). D, Functional classification of SIZ1-regulated genes. Genes whose expression levels were increased in siz1 under the Pi-sufficient (left panel) and Pi-deficient (right panel) conditions were classified based on their GO biological processes. WT, Wild type. [See online article for color version of this figure.]

Figure 8.

The expression levels of EXP17, GLH, and UGT73B4 were up-regulated in siz1 seedlings. Seedlings 3.5 d old were transferred onto medium that was supplemented with 1.25 mm (+Pi) or 0.0125 mm (−Pi) KH₂PO₄ and were incubated for 3 d. The relative mRNA levels were determined by quantitative RT-PCR analysis. White bars, wild type; black bars, siz1 mutant. The data shown are means ± sd (n = 3).

Figure 9.

The response to Pi deficiency is independent of SA signaling. The siz1-2 dwarf-like phenotype is suppressed by the expression of the nahG gene or the pad4 mutation (Lee et al., 2007). Wild-type (WT), siz1-2, pad4, nahG, siz1-2 pad4, and siz1-2 nahG seedlings at 3.5 d old that were grown on high-Pi medium were transferred onto medium containing 0.0125 mm KH₂PO₄. A, This photograph was taken 7 d after the transfer. B, The growth of PR was scored. All values shown represent means of 12 seedlings ± se. Bar = 10 mm. [See online article for color version of this figure.]

Figure 10.

A model illustrating that SIZ1 negatively regulates the Pi starvation-induced root system architecture modification through controlling the auxin accumulation. After Pi starvation (A), the plants somehow sense Pi starvation (B). Then, auxin is accumulated in the PR root tip, and PR elongation is inhibited (C). Auxin is then accumulated in the LR primordia and tips for elongation (D). Auxin accumulates more in younger LR tips than it does in older LR tips (E). After auxin accumulation in the LR tips cease (F), the plants are not able to survive (G). SIZ1 is involved in the negative regulation of auxin accumulation in the PR and LR tips (C–E). [See online article for color version of this figure.]