The protein quality control system manages plant defence compound synthesis

Abstract

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers1,2,3. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing3,4,5,6, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols7,8,9. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR10,11,12,13. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

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Figure 1: Expression of MKB1 and saponin synthesis genes is correlated.
Figure 2: MKB1 silencing causes the ‘makibishi’ phenotype.
Figure 3: MKB1 silencing causes ectopic accumulation of monoglycosylated saponins.
Figure 4: Mkb1 targets the HMGR enzyme.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The GenBank EMBL/DDBJ accession number for MKB1 is JF714982.

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Acknowledgements

We thank W. Ardiles-Diaz, S. Carbonelle, R. Dasseville, R. De Rycke and L. Ingelbrecht for technical assistance, and R. Dixon, H. Ezura, R. Hampton, A. Stolz and D. Wolf for providing plant and yeast materials. This research has received funding from the Agency for Innovation by Science and Technology in Flanders (‘Strategisch Basisonderzoek’ Combiplan project SBO040093), the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement number 222716 –SMARTCELL and the Spanish Ministerio de Economía y Competitividad under grant BFU2011-24208. T.M. and N.D.G. are indebted to the VIB International PhD Fellowship Program and the Agency for Innovation by Science and Technology for predoctoral fellowships, respectively. J.P. and S.L. are postdoctoral fellows of the Research Foundation Flanders (FWO).

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J.P., T.M., M.G.-G., N.D.G., S.L., R.V.B., P.M., A.K., C.J.G., A.T., W.O., N.C. and A.G. performed experiments and analysed the results. J.P., T.M., M.G.-G., N.D.G., S.L., K.M., C.J.G., S.G., N.C. and A.G. designed experiments and analyses. J.P., T.M., J.M.T. and A.G. wrote the manuscript. All authors commented on the results and the manuscript.

Corresponding author

Correspondence to Alain Goossens.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The protein quality control system manages plant defence compound synthesis in the model legume M. truncatula.

a, Summarizing schematic. The model depicts three cellular contexts in M. truncatula roots in which distinct ERAD-mediated control of HMGR activity occurs and the consequences thereof on root development (inset picture) and triterpene biosynthesis (sterols and glycosylated triterpene saponins (GTS)). The three conditions are (1) control roots cultured in control conditions (CTR; left) with normal ERAD survey of HMGR; (2) control roots cultured in the presence of jasmonate (+JA; middle) with increased triterpene saponin synthesis and increased ERAD activity; and (3) the Mkb1KD mutant roots (right) with reduced ERAD control of HMGR activity, leading to accumulation of bioactive monoglycosylated triterpene saponins. Dotted lines represent the endoplasmic reticulum. Arrows indicate flux through the pathway. Red colours reflect changes in comparison to the CTR condition. IPP, isopentenyl diphosphate. b, Schematic overview of the topology of HMGR enzymes and RMA- and HRD-type E3 ubiquitin ligases from yeast, M. truncatula and humans. c, Kyte–Doolittle hydropathy plot of S. cerevisiae Hmg2 (left), M. truncatula Hmgr1 (middle) and H. sapiens HMGCR (right), with window size 15. d, Kyte & Doolittle hydropathy plot of S. cerevisiae Hrd1 (left), M. truncatula Mkb1 (middle) and H. sapiens GP78 (right), with window size 15. Red bars indicate the hydrophobic transmembrane domains. GenBank accession numbers: H. sapiens: GP78, Q9UKV5; HMGCR, AAH33692; M. truncatula: Mkb1, JF714982; Hmgr1, ABY20972; S. cerevisiae: Hmg2, DAA09750; Hrd1, CAA99012.

Extended Data Figure 2 The triterpene saponin biosynthesis pathway in M. truncatula.

HMG, 3-hydroxy-3-methylglutaryl; P450, cytochrome P450.

Extended Data Figure 3 Sequence and structural analysis of eukaryotic RMA proteins.

a, Phylogenetic analysis of Mkb1 and other RMA-type E3 ubiquitin ligases. The percentage of replicate trees that clustered together in the bootstrap test is shown next to the branches. The scale bar indicates the number of amino acid substitutions per site. Arabidopsis thaliana (At), Capsicum annuum (Ca), Caenorhabditis elegans (Ce) and Homo sapiens (Hs) amino acid sequences were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Amino acid sequences of M. truncatula Mkb1 and homologous proteins (prefix TC) were retrieved from the Medicago truncatula Gene Index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb = medicago) following BLAST searches. b, Comparison of the amino acid sequence of Mkb1 with that of RMA proteins from A. thaliana, C. annuum, C. elegans and H. sapiens. Conserved amino acids that are identical in the seven proteins are indicated with an asterisk.

Extended Data Figure 4 The ‘makibishi’ phenotype.

a, CTR, Mkb1OE and Mkb1KD roots grown on solid medium. b, Confocal microscopy analysis of CTR, Mkb1OE and Mkb1KD roots grown in liquid medium. c, MKB1 transcript levels in transgenic M. truncatula hairy roots. y axis, the expression ratio relative to the normalized transcript levels of CTR line 1 in log scale. Error bars, ± s.e.m. (n = 3). Statistical significance was determined by Student’s t-test (**P < 0.01, ***P < 0.001). d, e, PCA (d) and PLS-DA (e) of samples from Mkb1KD (red), Mkb1OE (blue) and CTR (black) roots. f, LC-ESI-FT-ICR-MS chromatograms of seven saponin standards (the identity of which is indicated in g and numbered from 1 to 7), an extract of CTR roots, and an extract of Mkb1KD roots (from left to right). The coloured overlay chromatograms depict mass range scans, using a mass window of 0.01 Da, corresponding to the seven standards. g, MS2 fragmentations of the standards (black, top) compared to the fragmentation of the corresponding peaks in a CTR root extract (coloured, bottom). The numbers correspond to the numbers of the standards depicted in f.

Extended Data Figure 5 Phenocopy of the Mkb1KD phenotype.

a, LC-ESI-FT-ICR-MS chromatograms of the medium from CTR (black) and Mkb1KD (red) roots. The peak at tR 27.95 min represents 3-O-Glc-medicagenic acid. b, Light microscopy analysis of CTR hairy roots incubated for 1 week in medium supplemented with medium from CTR (left) or Mkb1KD (right) roots.

Extended Data Figure 6 Sterol synthesis in transgenic M. truncatula hairy roots.

a, Schematic overview of the sterol biosynthesis pathway. b, c, qRT–PCR analysis of sterol biosynthetic genes in CTR, Mkb1OE and Mkb1KD roots. y axis, the expression ratio relative to the normalized transcript levels of CTR line 3 in log scale. SQE, squalene epoxidase; SQS, squalene synthase. d, Sterol levels in CTR, Mkb1OE and Mkb1KD roots. y axis, sterol accumulation relative to the CTR lines. Error bars, ± s.e.m. (n = 3). Statistical significance was determined by Student’s t-test (*P < 0.1, **P < 0.01).

Extended Data Figure 7 Mkb1 has auto-ubiquitination activity and is an endoplasmic-reticulum-localized protein that associates with HMGR proteins.

a, Schematic representation of the Mkb1 protein and its domain structure. b, In vitro auto-ubiquitination assay of Mkb1. The bacterially expressed GST–MKB1 constructs were incubated with ATP in the presence or absence of His-tagged ubiquitin (His-UBQ), E1 (rabbit UBE1) and E2 (human UBCH5A). Samples were resolved by 8% SDS–PAGE, followed by protein immunoblot analysis with anti-GST (top) or anti-His (bottom) antibodies. The recombinant, truncated version of the Mkb1 protein, lacking the membrane anchor domain (Mkb1ΔC), possesses self-ubiquitination activity, whereas a mutated ‘ligase-dead’ version of the recombinant Mkb1ΔC protein, in which the essential amino acid residues Cys 37 and Cys 40 were substituted by Ser residues, does not. c, Subcellular localization of Mkb1 in bombarded onion cells. The pictures show the GFP signal and the GFP-brightfield merged image (left and right, respectively) of GFP–Mkb1 and GFP–Mkb1ΔC (top and bottom, respectively). The GFP–Mkb1 protein is visible in a network pattern whereas the GFP–Mkb1ΔC protein shows cytosolic localization. d, Subcellular localization of Mkb1 in yeast cells. The pictures show the signal of GFP–Mkb1 (left), Sec13–tagged to red fluorescent protein (RFP) (middle), and the merged image (right), respectively. e, Total protein lysates (TL, top panels) of M. truncatula roots producing GS-tagged versions of Mkb1 or the control proteins Jaz1 (a transcriptional repressor) and Cks1 (a cell cycle control protein) were immunoprecipitated with human IgG Sepharose beads (IP, bottom panels) and subjected to immunoblot analysis with the polyclonal antibodies raised against melon HMGR proteins. In total, association of GS-tagged Mkb1, Jaz1 and Cks1 proteins with HMGR was detected in 7 on 9, 2 on 4, and 0 on 3 independent experiments, respectively.

Extended Data Figure 8 HMGR levels and activity are altered in Mkb1KD lines.

a, Top, immunoblot analysis with polyclonal antibodies raised against melon (top) and Arabidopsis (bottom) HMGR proteins. Bottom, the fold induction in Mkb1KD lines relative to the control lines. Error bars, ± s.e.m. (n = 3). Statistical significance was determined by Student’s t-test (*P < 0.1). b, Specific HMGR activity in M. truncatula roots relative to the activity in CTR line 1 in log scale. c, The stability of HMGR–firefly luciferase (fLUC) fusion proteins in MKB1 (+M)-transfected tobacco protoplasts relative to the fLUC value measured in the absence of MKB1 (−, set at 100%). Error bars, ± s.e.m. (n = 24). Statistical significance was determined by Student’s t-test (** P < 0.01).

Extended Data Figure 9 Deregulated HMGR activity causes the ‘makibishi’ phenotype.

a, CTR and tHmgr4OE hairy roots grown on solid medium. b, Scanning electron microscopy analysis of CTR, Mkb1KD and tHmgr4OE roots grown on solid medium. Scale bar, 250 μm. c, d, Three-dimensional serial block-face-scanning electron microscopy image stacks visualizing the cell structures of tHmgr4OE roots grown on solid medium. IMOD, FIJI and Ilastik software were used to generate orthogonal slices (c) and three-dimensional reconstructions (d). Yellow lines indicate positions of the corresponding orthogonal views. Scale bar, 10 μm. e, Light microscopy analysis of CTR hairy roots maintained for 4 weeks on medium supplemented with increasing amounts of lovastatin (in μM). f, g, Expression analysis of tHmgr4OE lines. f, (t)HMGR4 transcript levels in tHmgr4OE roots. The different panels respectively show PCR with reverse transcription (RT–PCR) analysis of the GFP (control) and tHMGR4 transgene transcript levels only (left), qRT–PCR analysis of the endogenous HMGR4 transcript levels only (middle) and qRT–PCR analysis of total HMGR4 transcript levels (transgene and endogene; right). g, qRT–PCR analysis of saponin biosynthetic genes in tHmgr4OE and Mkb1KD roots. y axis, the expression ratio relative to the normalized transcript levels of CTR line 3 in log scale. Error bars, ± s.e.m. (n = 3). Statistical significance was determined by Student’s t-test (*P < 0.1, **P < 0.01, ***P < 0.001). h, Accumulation of monoglycosylated saponins in tHmgr4OE and Mkb1KD roots. Average total ion current of the peaks corresponding to soyasaponin I (left) and 3-O-Glc-medicagenic acid (right). TH, tHmgr4OE roots. Error bars, ± s.e.m. (n = 3). i, Immunoblot analysis for 6myc-tagged Hmg2 and Coomassie blue staining (top and bottom, respectively) of protein extracts from HRD1 (H) or hrd1 (h) yeast cells transformed with MKB1 (+M) or a ligase-dead version (+m). The destination vector pAG426GPD was used as a control (−).

Extended Data Table 1 LC-ESI-FT-ICR-MS analysis of M. truncatula hairy roots

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Pollier, J., Moses, T., González-Guzmán, M. et al. The protein quality control system manages plant defence compound synthesis. Nature 504, 148–152 (2013). https://doi.org/10.1038/nature12685

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