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PDK1 regulates auxin transport and Arabidopsis vascular development through AGC1 kinase PAX

Nature Plants volume 6, pages 544–555 (2020)Cite this article

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Abstract

The 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a conserved master regulator of AGC kinases in eukaryotic organisms. pdk1 loss of function causes a lethal phenotype in animals and yeasts, but only mild phenotypic defects in Arabidopsis thaliana (Arabidopsis). The Arabidopsis genome contains two PDK1-encoding genes, PDK1 and PDK2. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate true loss-of-function pdk1 alleles, which, when combined with pdk2 alleles, showed severe developmental defects including fused cotyledons, a short primary root, dwarf stature and defects in male fertility. We obtained evidence that PDK1 is responsible for AGC1 kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX) activation by phosphorylation during vascular development, and that the PDK1 phospholipid-binding Pleckstrin Homology domain is not required for this process. Our data indicate that PDK1 regulates polar auxin transport by activating AGC1 clade kinases, resulting in PIN phosphorylation.

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Fig. 1: Arabidopsis pdk1 pdk2 loss-of-function mutants exhibit a dwarf stature, but do not phenocopy pid mutants.
Fig. 2: The phenotype and auxin response pattern of pdk1 pdk2 mutant seedlings resembles that of auxin transport inhibitor-treated seedlings.
Fig. 3: PDK1 is not involved in PIN polarity control.
Fig. 4: PDK1-dependent activation of PAX directs protophloem differentiation.
Fig. 5: The unique pdk1-13 pdk2-4 mutant cotyledon vein pattern can be partially rescued by PDK1S0 and PAX(SD).
Fig. 6: Alternative splicing produces a PDK1 transcript encoding a functional PDK1 variant lacking the PH domain.
Fig. 7: The PH domain is not essential for PDK1 function in the auxin-sensing rheostat during vascular development.

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Data availability

All processed data are contained either in the manuscript, Extended Data or Supplementary Information. Raw data and materials generated during this study are available upon reasonable request.

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Acknowledgements

We thank A. Rodriguez-Villalon, C. Hardtke, C. Schwechheimer, I. Heilmann and J. Chang (Jia Li’s laboratory) for providing mutants cvp2 cvl1, pax paxl, d6pk012, pip5k1 pip5k2 and gnom, respectively. We thank T. Devarenne for providing yeast strain p416GPD, S. de Pater for providing the CRISPR/Cas9 plasmids, X. Men for sharing preliminary data on PIN2HL phosphorylation by PDK1, K. Boot for help with auxin transport assays and G. Lamers and J. Willemse for help with microscopy. We are grateful to N. Surtel, W. de Winter and J. Vink for their help with plant growth and media preparation. This project was supported by the China Scholarship Council.

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  1. Yao Xiao

    Present address: Plant Systems Biology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany

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  1. Plant Developmental Genetics, Institute of Biology Leiden, Leiden University, Leiden, the Netherlands

    Yao Xiao & Remko Offringa

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Y.X. and R.O. conceived the project, designed experiments and analysed and interpreted results. Y.X. performed the experiments and R.O. supervised the project. Y.X. and R.O. wrote the manuscript.

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Extended data

Extended Data Fig. 1 Relevance of PDK1 for PID function in planta.

a–g, Representative 7-day-old seedlings of the indicated lines. Please note that only p35S::PID#21 seedlings show agravitropic growth. Scale bars represent 1 cm. Similar results have been obtained in four independent experiments. h, Box plot showing the quantification of the primary root length of 7-day-old seedlings of Arabidopsis wild type (Col-0), p35S::PDK1 lines #3.7 and 13.3 (red box), p35S::YFP:PDK1 lines #1.5, 5.4 and 9.6 (blue box), and p35S::PID line #21. n indicates the number of measured roots, the results are from a single experiment, but similar results were obtained in three independent experiments. Lower case letters indicate statistically different groups (p < 0.05, details of statistical analysis are provided in Supplementary Table 4), as determined by a one-way ANOVA followed by Tukey’s test. Boxes indicate 1st and 3rd quartile, the horizontal line in a box the median, whiskers the maximum and minimum. i, PDK1 expression levels in Col-0 and in the p35S::PDK1 and p35S::YFP:PDK1 lines used in h. The bar graph shows the mean value ± SEM (n = 3 technical repeats). j, Representative images showing a detail of the root tip phenotype of seedlings in a–h. White arrow heads point out collapsed (p35S::PID#21) or normal root meristems (all other lines; n = 40 independent seedling roots were checked for each line). Scale bars indicate 0.5 mm. k, One-month-old Col-0 and PDK1 overexpression plants (n = 10 independent plants were observed per line). l, Inflorescence phenotype of Col-0, PID- and PDK1 overexpression lines (n = 12 independent plants were observed per line). m, Representative images of PID:YFP subcellular localization in Col-0 or pdk1-13 pdk2-4 protoplasts. Eleven wild-type (Col-0) and fourteen pdk1-3 pdk2-4 mutant protoplasts were observed in this experiment, and all showed the similar localization. For h-m, Three biological repeats showed similar results.

Extended Data Fig. 2 Quantification of pdk1 pdk2 mutant phenotypes and complementation of the mutant by 35S::PDK1 or 35S::YFP:PDK1.

a, Quantification of the rosette diameter of 30-day-old Arabidopsis wild-type (Col-0) and pdk1-13 pdk2-4 plants. A two-sided Student t-test was used for statistical analysis. b, Final heights of the indicated plant lines. c, Flowering time of the indicated lines. Lowe case letters in b and c indicate statistical differences, as determined by one-way analysis of variance (ANOVA) followed by Tukey’s test (p < 0.05, details of statistical analysis are provided in Supplementary Table 4). n indicates the number of independent plants used for measurements in a-c. Boxes indicate 1st and 3rd quartile, the horizontal line in a box the median, whiskers the maximum and minimum. d-k, Plants of wild-type (Col-0), pdk1-13 pdk2-4 and representative complementation lines were grown on plates for 10 days and subsequently in soil for 20 days before photographing. 15 plants were observed in a single experiment for each line. For a-k, three independent experiments showed similar results.

Extended Data Fig. 3 pdk1 pdk2 mutant phenotypes in reproductive tissues.

a, pdk1 pdk2 siliques (three on the right) are much shorter than wild-type siliques (three on the left), and contain many unfertilized ovules. b and c, Difference in pollen grain deposition on the stigma of a wild-type (b) or a pdk1-13 pdk2-4 mutant (c) flower. d and e, Mature wild-type (d) or pdk1-13 pdk2-4 mutant (e) anthers stained with Alexander’s showing that pollen grains are viable, but that mutant anthers do not sufficiently dehisce. f and g, In vitro germination of wild-type (f) and pdk1-13 pdk2-4 mutant (g) pollen. A detail of pollen tube tip is shown in the inset. h, Relative pollen tube length after 18 hours incubation. The average length of wild-type (Col-0) pollen tubes is put at 1.0. A two sided Student’s t-test was used for statistical analysis, n indicates the number of independent pollen tubes measured. Means with 95% confidence intervals are shown. i, Developmental series of pdk1-13 pdk2-4 mutant and wild-type (Col-0) flowers. The white bars indicate the position of the gynoecium apex, the yellow bars indicate the position of the anthers. j, Ripe siliques with the valves removed, derived from reciprocal crosses between wild-type Arabidopsis (Col-0) and the pdk1-13 pdk2-4 loss-of-function mutant. k and l, Representative DIC images showing the phenotype of wild-type (Col-0) (k, n = 330) or pdk1-14 pdk2-4 mutant (l, n = 306) ovules. For a–l, similar results were obtained in three independent experiments.

Extended Data Fig. 4 pdk1 pdk2 mutant seedlings phenocopy d6pk012 triple mutants.

a, Phenotype of 15-day-old wild-type (Col-0) and pdk1-11 pdk2-1 seedlings grown on vertical plates. b, Apical hook phenotype of 4-day-old wild-type (Col-0) and pdk1-13 pdk2-4 etiolated seedlings. c, Hypocotyl curvature after 18 h directional white light treatment on 4-day-old etiolated wild-type (Col-0) and pdk1-13 pdk2-4) seedlings (n = 25 independent seedlings). A two sided Student’s t-test was used for statistical analysis. Means with 95% confidence intervals are shown. d, 5-day-old wild-type (Col-0) and pdk1-13 pdk2-4 seedlings grown in dark. Note the agravitropic hypocotyl growth of pdk1-13 pdk2-4 mutant seedlings. For a and d, five independent experiments showed similar results. For b and c, three independent experiments showed similar results.

Extended Data Fig. 5 PDK1 and PDK2 are predominantly expressed in (pro) vascular tissues, where PDK1 associates with the basal PM.

a–j, PDK1 expression is confined to the provascular tissue during Arabidopsis embryo development. Confocal (a–e), and bright field images (f-j) of Arabidopsis pPDK1::YFP:PDK1 16-cell (a, f), globular (b, g), heart (c, h), late heart (d, i), and torpedo (e, j) stage embryos. k, Confocal images of a 4-day-old pPDK1::YFP:PDK1 root tip. The inset shows a detail of YFP:PDK1 localization in root stele cells. l-u, Spatio-temporal expression pattern of PDK1 (l, m, n, r, s) and PDK2 (p, q, o, t, u) as reported by histochemical staining of 3-day-old seedlings (l, p), 7-day-old seedlings (m, q), 16-day-old plants (n, o) and inflorescences and siliques from 40-day-old plants (r-u) of representative pPDK1-GG and pPDK2-GG lines, respectively. Similar results were obtained with three independent lines in three biological repeats.

Extended Data Fig. 6 PDK1 strongly phosphorylates the PIN2 central hydrophilic loop (PIN2HL) in a S1, S2, S3-independent manner in vitro.

Only very weak (probably non-specific) phosphorylation is observed for the PIN1HL, PIN3HL or PIN7HL. Red or green arrows point out the position of the GST-PINHL or GST-PDK1, respectively. Upper: autoradiograph, lower: PageBlue stained gel. Similar results were obtained in three independent experiments.

Extended Data Fig. 7 PDK1 and PDK2 are expressed in primary root vascular tissue to control phloem differentiation.

a, b, DIC microscopy images of roots of 5-day-old PDK1 (a) and PDK2 (b) promoter::turboGFP-GUS seedlings stained for GUS activity. Bar = 0.1 mm. c, d, Confocal sections of mPS-PI stained wild-type (Col-0, c) and pdk1-13 pdk2-4 (d) roots. The protophloem cell layer is indicated with a red asterisk. Bar =10 μm. e, Quantification of gaps in PPSEs in wild-type and pdk1-13 pdk2-4 roots. The number of observed roots (n) is indicated in each bar. Three biological repeats showed similar results.

Extended Data Fig. 8 Complementation of the pdk1 pdk2 mutant by PDK1 promoter driven YFP:PDK1, YFP:PDK1S0 or YPF:PAX(SD) expression.

a–o, Phenotype of 13-day-old seedlings of the indicated lines grown on vertical plates. pdk1 pdk2 pPDK1::YFP- is omitted in (d–o) for presentation purposes. j–l, Note that YFP:PAX only shows partial complementation in the high expressing line #4.7 (see also Fig. 4a). Five biological repeats showed similar results. p and q, pPDK1::YFP:PAX (SD) partially rescues the dwarf rosette phenotype of pdk1-14 pdk2-4 (q), whereas pPDK1::YFP:PAX does not (p). Rosette phenotype of 26-day-old wild-type (Col-0), pdk1-14 pdk2-4 mutant, or pPDK1::YFP:PAX pdk1-14 pdk2-4 (p) or pPDK1::YFP:PAX pdk1-14 pdk2-4 (q) plants. For the latter two, representative plants of nine independent lines are shown. Similar results were obtained in the T2, T3 and T4 generation.

Extended Data Fig. 9 PDK1 promoter-driven expression of the cytosolic PDK1S0 isoform rescues the pdk1 pdk2 mutant rosette phenotype.

a, Complementation assay for overexpression of cDNAs representing the different PDK1 splice variants in the pdk1 pdk2 mutant background. Plants were grown on plates for 10 days, and subsequently transferred to and grown in soil for 10 days. Similar results were obtained in the T2, T3 and T4 generation. b and c, Subcellular localization of PDK1 promoter driven YFP:PDK1 (b, the line with lowest expression level obtained) and YFP:PDK1S0 (c, the line with highest expression level obtained) in root columella cells. Ten roots were observed per independent experiment for each line. d, PDK1S0 (lower panel) has a higher auto- and trans-phosphorylation activity compared to PDK1 (upper panel). Coomassie stained gel (left) and autoradiograph (right) are shown, and positions of the GST-tagged PAX, -PDK1 and -PDK1S0 are indicated. For b–d, similar results have been obtained in three independent experiments.

Extended Data Fig. 10 PDK1 expression and YFP:PDK1 subcellular localization after chemical treatment.

a, Relative PDK1 transcript levels are not significantly changed in 5-day-old seedlings following 0.5 to 4 hours treatment with 1 μM indole-3-acetic acid (IAA). Bars with error bars indicate mean ± SEM (n = 3 technical repeats). b–e, Confocal images of pdk1 pdk2 pPDK1::YFP:PDK1 roots showing YFP:PDK1 localization after 1 hour treatment with DMSO (b), 1 μM IAA (c) or 33 μM wortmannin (WM) (d), or 30 min treatment with 30 μM phenylarsine oxide (PAO) (e). For a–e, Similar results have been obtained in three biological repeats.

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Xiao, Y., Offringa, R. PDK1 regulates auxin transport and Arabidopsis vascular development through AGC1 kinase PAX. Nat. Plants 6, 544–555 (2020). https://doi.org/10.1038/s41477-020-0650-2

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