INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-dependent inositol polyphosphates regulate auxin responses in Arabidopsis

Abstract The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOG 1 (ASK1), and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants.


Supplemental
. Thermomorphogenic responses and primary root growth are controlled by ITPK1. (A) Represenatitive plate pictures of designated genotypes grown on solidified half-strength MS media supplemented with 1% sucrose under control condition or at higher temperature. 5-dayold seedlings were kept at 22°C or shifted to 29°C and kept for 8 daysbefore picture was taken. (B) Relative root length of wild-type (Col-0) and itpk1 mutant treated with 100 nM 1naphthaleneacetic acid (NAA). Seeds of indicated genotypes were surface sterilized and sown on sterile solidified half-strength MS media supplemented with 1% sucrose with or without NAA. Germinated seedlings were allowed to grow for 16 days. Root lengths were evaluated by ImageJ. Letters depict significance in a one-way ANOVA followed by Tukey's test (a and b, P < 0.001; b to c, P < 0.001; a to c, P < 0.01). Data are shown as means ± SEM, n=10-29.
Supplemental Figure S4. Auxin-related growth and developmental processes are not affected in Arabidopsis pho2-1 plants.
(A) Root gravitropism of seedlings of wild-type (Col-0) and pho2-1 mutant after 90° reorientation. 7-day-old seedlings of Col-0 and pho2-1 were transferred to solidified halfstrength MS media supplemented with 1% sucrose and after another 12 days of growth, the seedlings were rotated by 90° and the gravitropic curvature was measured after 16 h. The distribution of data was analyzed using a χ2 test (number of seedlings n ≥ 22, groups contained at least 4% of total seedlings per genotype). Same letter (a) denotes that no significant differences at P < 0.05 were detected. (B) Effect of auxin on the primary root length of the phosphorus overaccumulator mutant pho2-1. 6-day-old seedlings of wild-type (Col-0) and pho2-1 were transferred to solidified halfstrength MS media supplemented with 1% sucrose and with 0 or 25 nM indole-3-acetic acid (IAA) and 625 μM phosphate. Primary root length was measured 7 days after transferring plants to treatments. Bars show means ± SEM (n = 11) . No significant differences at P < 0.05 were detected by two-tailed Student's t-test. (C) 5-day-old seedlings of wild-type (Col-0) and pho2-1 grown at 22°C grown on solidified halfstrength MS media supplemented with 1% sucrose were kept at 22°C or shifted to 29°C. Root length was evaluated after 8 days by ImageJ. Error bars represent SEM, n ≥ 23. No significant differences at P < 0.05 were detected by two-tailed Student's t-test. Figure S5. Role of ITPK1 in phosphorus accumulation and the effect of auxin.

Supplemental
(A-C) Shoot phosphorous (P) concentration and relative primary root lengths of wild-type (Col-0) and itpk1mutant grown under increasing indole-3-acetic acid (IAA) concentrations. DW denotes dry weight. 6-day-old seedlings were transferred to solidified half-strength MS media with 1% sucrose supplemented with 0, 25 and 75 nM IAA under 625 µM phosphate (High P) (A and B) or 10 µM phosphate (Low P) (C). Shoot phosphorus concentration (A) and relative primary root length (B and C) were assessed 7 days after transferring plants to treatments. Bars show means ± SEM (n = 4 replicates with 4 plants each for shoot phosphorus analysis and 15 individual plants for primary root length). Absolute values for phosphorus concentrations were compared by ANOVA and post-hoc Tukey test and different letters indicate significant differences at P < 0.05. Relative root growth was compared by pairwise Student's t-test and significant differences at P < 0.01 are indicated by single asterisk. Figure S6. VIH2-deficient plants are not compromised in auxin perception.

Supplemental
(A) Root gravitropism of seedlings of wild-type (Col-0), vih2-3 and vih2-4 mutants after 90° reorientation. 7-day-old seedlings of indicated genotypes grown on solidified half-strength MS media supplemented with 1% sucrose, were transferred to new solid media and after another 12 days of growth, the seedlings were rotated by 90° and the gravitropic curvature was measured after 16 h. The distribution of data was analyzed using a χ2 test (number of seedlings n ≥ 35). Same letter denotes that no significant differences at P < 0.05 were detected. The experiment was repeated independently with similar results. (B) Relative root length of wild-type (Col-0), vih2-3 and vih2-4 mutants treated with 100 nM indole-3-acetic acid (IAA). Seeds were surface sterilized and sown on sterile solidified halfstrength MS media supplemented with 1% sucrose. 6-day-old seedlings were transferred to new media containing either 100 nM IAA or DMSO as control and scanned after 7 days. Root lengths were evaluated by ImageJ. Data are means ± SEM, n≥34. No significant differences at P < 0.05 were detected by two-tailed Student's t-test. The experiment was repeated independently with similar results. (C) Primary root length analysis of seedlings of wild-type (Col-0), vih2-3 and vih2-4 mutants grown at higher temperatures. 5-day-old seedlings of designated genotypes were grown on solidified half-strength MS media supplemented with 1% sucrose at 22°C, then kept at 22°C or shifted to 29°C. Root length was evaluated after 8 days by ImageJ. Error bars represent SEM, n ≥ 25. No significant differences at P < 0.05 were detected by two-tailed Student's t-test. The experiment was repeated independently with similar results.
Supplemental Figure S7. The ipk1-1 mutant is defective in auxin perception and InsP/PP-InsP homeostasis.
(A) Root gravitropism of seedlings of wild-type (Col-0) and ipk1-1 mutant after 90° reorientation. 12-day-old seedlings of indicated genotypes grown on solidified half-strength MS media supplemented with 1% sucrose were rotated by 90° and the gravitropic curvature was measured after 16 h. The percentage of the seedlings in each category is represented by the length of the bar. FW denotes fresh weight. The distribution of data was analyzed using a χ2 test (number of seedlings n ≥ 20). Means with different letters are significantly different, P < 0.005. The experiment was done independently with similar results.
(C) CE-ESI-MS analysis of inositol polyphosphate levels of shoots of 35-day-old Arabidopsis wild-type (Col-0) and the ipk1 mutant. Plants were cultivated in hydroponics with sufficient supply of all nutrients. Data are means ± SEM (n = 3 biological replicates). *P < 0.05, **P < 0.01 and ***P < 0.001 , according to two-tailed Student's t-test (ipk1 vs Col-0). The same Col-0 extracts used in this analysis also served as control in a previous study (Riemer et al., 2021). Col-0 and ipk1 plants were grown together in the same experiment and samples harvested, extracted and analyzed at the same time. InsP denotes inositol phosphate.
Supplemental Figure S8. Binding of InsPs to the auxin-receptor complex.

(A) SAX-HPLC of ITPK1 kinase reaction. [ 3 H]-InsP4a was purified from [ 3 H] inositol-labeled itpk1-2 plants and incubated with recombinant ITPK1 and ATP. The kinase product was resolved by SAX-HPLC. InsP denotes inositol phosphate. (B) Direct binding of [ 3 H]-InsP3b, [ 3 H]-InsP4a, and [ 3 H]-InsP6 to the TIR1/ASK1/IAA7 auxin receptor complex. A total activity of 2000 cpm was used for each [ 3 H]-labeled InsP species. [ 3 H]-InsP3b and [ 3 H]-InsP4a were purified and desalted from [ 3 H]-myo-inositol labeled seedlings of the itpk1-2 mutant and [ 3 H]-InsP6 from Col-0 seedlings. Values show means ± SEM (n = 2). InsP denotes inositol phosphate.
Supplemental Figure S9. The itpk2-2 lines are not defective in InsP synthesis and auxin responses. (A) The itpk2-2 line appears not to be compromised in InsP synthesis. Extracts of designated [ 3 H] inositol-labeled Arabidopsis seedlings were resolved by SAX-HPLC. Activities obtained by scintillation counting of fractions containing the InsP2-InsP8 peaks are presented. (B) Root gravitropism of seedlings of wild-type (Col-0) and itpk2-2 plants after 90° reorientation. 7-day-old seedlings grown on solidified half-strength MS media supplemented with 1% sucrose were transferred to new media and after another 12 days of growth, the seedlings of Col-0 and itpk2-2 were rotated by 90° and the gravitropic curvature was measured after 16 h. The distribution of data was analyzed using a χ2 test (number of seedlings n ≥ 35). No significant differences at P < 0.05 were detected. (C) Relative root length of wild-type (Col-0) and itpk2-2 mutant treated with 100 nM indole-3acetic acid (IAA). Seeds of indicated genotypes were surface sterilized and sown on sterile solidified half-strength MS media supplemented with 1% sucrose. 6-day-old seedlings were transferred to new plates containing either 100 nM IAA or DMSO as control and scanned after 7 days. Root lengths were evaluated by ImageJ. Data are means ± SEM, n=37. The experiment was repeated independently with similar results. No significant differences at P < 0.05 were detected by two-tailed Student's t-test. Figure S10. ITPK1 and ITPK2 act redundantly to control root gravitropism.

Supplemental
Root gravitropism of wild-type (Col-0), itpk1, itpk2 and itpk1itpk2 double knockout plants. 7day-old seedlings grown on solidified half-strength MS media supplemented with 1% sucrose were transferred to new media and after another 7 days of growth, the seedlings of indicated genotypes were rotated by 90° and the gravitropic curvature was measured after 28 h.

Supplemental Tables
Supplemental Table S1. Primer list.
Primer list for PCR-based characterization of T-DNA insertion lines.