The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses

Abstract

Plants sense phosphate (Pi) deficiency and initiate signaling that controls adaptive responses necessary for Pi acquisition. Herein, evidence establishes that AtSIZ1 is a plant small ubiquitin-like modifier (SUMO) E3 ligase and is a focal controller of Pi starvation-dependent responses. T-DNA insertional mutated alleles of AtSIZ1 (At5g60410) cause Arabidopsis to exhibit exaggerated prototypical Pi starvation responses, including cessation of primary root growth, extensive lateral root and root hair development, increase in root/shoot mass ratio, and greater anthocyanin accumulation, even though intracellular Pi levels in siz1 plants were similar to wild type. AtSIZ1 has SUMO E3 ligase activity in vitro, and immunoblot analysis revealed that the protein sumoylation profile is impaired in siz1 plants. AtSIZ1-GFP was localized to nuclear foci. Steadystate transcript abundances of Pi starvation-responsive genes AtPT2, AtPS2, and AtPS3 were moderate but clearly greater in siz1 seedlings than in wild type, where Pi is sufficient. Pi starvation induced the expression of these genes to the same extent in siz1 and wild-type seedlings. However, two other Pi starvation-responsive genes, AtIPS1 and AtRNS1, are induced more slowly in siz1 seedlings by Pi limitation. PHR1, a MYB transcriptional activator of AtIPS1 and AtRNS1, is an AtSIZ1 sumoylation target. These results indicate that AtSIZ1 is a SUMO E3 ligase and that sumoylation is a control mechanism that acts both negatively and positively on different Pi deficiency responses.

Fig. 1.

Arabidopsis plants harboring siz1 mutations are hyperresponsive to Pi limitation in root architecture development. (A) Photographs are of representative wild-type [Col-0 or Col-0 gl1 (Col-gl1)], sos3-1, sos3-1 siz1-1, siz1-2, and siz1-3 seedlings 11 days after transfer onto medium containing 1/20× MS macronutrients without or with KH₂PO₄ supplement. sos3-1 and sos3-1 siz1-1 are in the Col-0 gl1 background, and siz1-2 and siz1-3 are in the Col-0 background. (B) Photographs are of wild-type and siz1-2 seedling roots 7 days after transfer onto medium containing 1/20× MS macronutrients without (–Pi) or with 1.25 mM KH₂PO₄ (+Pi).

Fig. 2.

Pi_int in the shoot and the root of wild-type and siz1 seedlings and plants is comparable. Shown are seedlings grown for 11 days on medium containing 1/5× MS macronutrients with 0.0125 or 1.25 mM KH₂PO₄ (A) or plants grown for 2 weeks in hydroponic solution with 0.01 or 1 mM KH₂PO₄ (B) were harvested, mean values ± SE, n = 6. pho1 and pho2 seedlings were used as controls in A (8, 9).

Fig. 3.

AtSIZ1 locus is At5g60410 as determined by thermal asymmetric interlaced PCR and T-DNA insertion diagnostic PCR. (A) Gene organization of AtSIZ1 and schematic representation of T-DNA insertion alleles are illustrated (arrowheads). Filled boxes are exons, and open rectangles are 5′ or 3′ untranslated regions. (B) Diagram illustrates the domain organization of AtSIZ1 (see Results). (C) AtSIZ1 is expressed in the root (R) and the shoot (S) of the wild type but not in these organs of siz1 seedlings.

Fig. 4.

In vitro and in vivo assays indicate that AtSIZ1 is a SUMO E3 ligase. (A) In planta sumoylation profiles of wild-type and siz1-2 seedlings that were grown for 10 days on 1× MS medium at 24°C (24) and then exposed to a 30-min heat shock of 40°C (40). (B) Analysis of in vitro sumoylation profiles indicates that AtSIZ1 facilitates production of ScSmt3-ScCdc3 conjugates. The indicated amount of GST-AtSIZ1 (0, 2, 4, or 10 μg) or ScSIZ2 (0 or 2 μg) was added to the reaction mixture and incubated with 10 mM ATP at 37°C for 0 (inc. –) or 90 min (inc. +) as described in ref. . Immunoblot analysis was performed with anti-ScSmt3 (Upper) or anti-T7 (detecting T7-ScCdc3; Lower). Filled arrowheads indicate bands corresponding to ScSmt3-ScCdc3 conjugates. Open arrowheads identify the AtSIZ1-ScSmt3 and ScSiz2-ScSmt3 conjugates. An asterisk identifies the T7-ScSIZ2-His protein. Numerals identify the migration position and molecular mass of the markers (kilodaltons).

Fig. 5.

Pi-responsive transcript abundance is altered in siz1 plants, and AtSIZ1 facilitates sumoylation of PHR1. (A) Pi starvation-responsive gene mRNA levels in Col-0 (wild type, open bars) and siz1-2 (filled bars) seedlings were determined by quantitative PCR. Seedlings were grown in liquid medium containing 1/5× MS macronutrients and 1× MS micronutrients with 1.25 mM KH₂PO₄ for 7 days and then transferred to medium without KH₂PO₄. Samples were collected immediately after transfer to medium without Pi (0 h) or after various time points in this medium (6, 12, or 48 h). (B) AtSIZ1 mediates in vitro sumoylation of PHR1. T7-PHR1 or T7-PHR1(2KR) were used as substrates for sumoylation in the reaction mixture containing ScAos1 (E1), ScUba2 (E1), ScUbc9 (E2), ScSmt3 (SUMO, 23), and AtSIZ1 (E3) proteins. PHR1 proteins were detected with anti-T7 antibody. Arrowheads indicate the position of sumoylated PHR1 proteins.