The Discovery of Plant Natriuretic Peptides. Physiology, molecular biology, systems biology and biotechnology

Transcription

1 The Discovery of Plant Natriuretic Peptides Physiology, molecular biology, systems biology and biotechnology

2 ANP - ATRIAL NATRIURETIC PEPTIDE ANP is a 28 aa peptide hormone ANP is produced in cardiocytes The targets are the vascular and renal system Some ANP receptors have guanylate cyclase domains NPs play a key role in homeostasis

3 Can vertebrate NPs affect plants? Do plants have a NP-like system? If plants contain NPs, can they affect vertebrates?

4 Stomatal function is a key to homeostasis in higher plants Stomatal opening is achieved by H 2 O uptake preceded by net K + influx which in turn is driven by H + -ATPase-dependent hyperpolarisation. Net H 2 O efflux causes closure and requires net K + efflux usually resulting from depolarisation.

5 First evidence ANP promotes concentration dependent stomatal opening Therefore: ANP must cause net K + (and consequent H 2 O) up-take Second messengers include: - [Ca 2+ ] cyt - [ph] cyt - inositol-1,4,5-trisphosphates - cyclic nucleotides (cgmp)

6 Isolation and identification of Plant Natriuretic Peptides - PNPs

7 - Anti-ANP purifies PNPs a biologically active plant peptide - PNPs modulate H 2 O and ion transport at nanomolar concentrations - PNPs are the first peptidic plant hormones that affect plant homeostasis

8 Structural analysis of PNPs irpnp Marker upper band: p kd 9 kd N-terminus: determined by Edman degradation C-terminus: determined by Mass Spec. - The AA sequence shows a high degree of homology with a blight induced, functionally uncharacterised protein (p12) from citrus

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10 The molecular structure of PNP-like molecules

11 Citrus p12 MGVGTKVLVITTMAICLISSAAYASEGTATFYTPPYVPSACNGYKNDGVMIAAASYAIWN 60! AtPNP-B ---MSKSIVFFSTVLVFLFSFSYATPGIATFYTS-YTP--CYRGTQEGVMIAAASDTLWD! AtPNP-A --MAVKFVVVMIVFAQILAPIAEAAQGKAVYYDPPYTRSACYG-TQRETLVVGVKNNLWQ! * :*. ::. : *: * *.:*. *. *.:.::... :*:! Citrus p12 AtPNP-B AtPNP-A Citrus p12 AtPNP-B AtPNP-A NGAVCNKSFRVKCTGATNQGTPHPCRGGSVLVKIVDLCPAGCQATIDLSQEAFSQIANPD! NGRVCGKMFTVKCSGPRN-AVPHPCTGKSVKVKIVDHCPSGCASTLDLSREAFAQIANPV! NGRACGRRYRVRCIGATY-NFDRACTGRTVDVKVVDFCREPCNGDLNLSRDAFRVIANTD! **.*.: : *:* *. :.* * :* **:** * *. ::**::** ***.! AGKIKIEFNQA! AGIINIDYFP-! AGNIRVVYTP-! ** *.: :! R-arginine W-tryptophan AtPNP-A; Theoretical pi: 9.32 (and 9.35 without signal peptide)

12 35 68 AtPNP-A --- PYTRSACYGTQRETLVVGVKNNLWQNGRACGRRY --- α-hanp SLRRSSCFGGR--MDRIGAQSGLGCN----SFRY **:*:* : :*.:..* * ** AtPNP-A(35-68) is the biologically active domain of the molecule

13 C β2 β6 β5 β1 N ATPNP-A: MAVKFVVVMIVFAQILAPIAEAAQGKAVYY DPPYTRSACYGTQRETLVVGVKNNLWQNGR ACGRRYRVRCIGATYNFDRACTGRTVDVKV VDFCREPCNGDLNLSRDAFRVIANTDAGNI RVVYTP β3 β4 Six stranded β-barrel structure with two ψ loops

14 PNPs in an evolutionary context

15 Index 6 M. trunculata Index 8 G. max AtPNP-B A. thaliana PNP-A Index 5 M. trunculata Index 4 O. sativa CjBAp12 C. jambhiri PNP-B AtPNP-A A. thaliana Index 1 O. sativa Index 2 O. sativa Indices 3 & 7 O. sativa β expansin A. thaliana α expansin N. tabacum α expansin T. vesicolor β-expansin β expansin O. sativa β expansin A. lentiformis α expansin R. diphyllum α expansin O. sativa α expansin M. quadrifolia α expansin β expansin O. sativa 0. 1

16 Hypothesis: A highly expressed class of molecules that perform an extracellular function have been recruited for a role in homeostasis

17 N Intron Intron C Expansins GH45 domain Cellulose-binding domain N C PNP-like molecules - Expansins have no glucanase activity - Glucanases have no expansin activity - p12 (PNP) has no expansin activity - PNPs are mobile molecules

18 Ancestral molecule has hydrolytic activity on Wall substrate Loss of hydrolytic activity Intron Intron Intron N C N C GH45 domain CB domain GH45 domain - Wall remains the substrate - CB domain restricts mobility - Cell membrane is the substrate - Absence of a CB domain increases mobility Expansins PNPs Both classes of molecules are extra cytosolic & may cooperate in cell elongation growth

19 Defining the biological role of PNP-like molecules in planta

20 In situ localisation of PNPs in conductive tissue (a) (b) (c) Indication of systemic action

21 In situ tissue print with anti-hanp and anti-pnp A vb B ph pith x ep C vb D vb A. Cut through ivy petiole; x - xylem, ph - phloem, ep - epidermis, vb - vascular bundle B. Detail C. Incubation o/n with anti-hanp Ab D. Incubation o/n with anti-pnp Ab

22 PNP is localised in the conductive tissue PNP extracted from the xylem is biologicaly active The molecular structure and systemic action are compatible

23 We would expect NPs to affect ion transport Directly: e.g. by forming or modifying channels or affecting pumps Indirectly: By mobilizing 2 nd messengers that affect (gate) channels or affect pumps

24 Time course of PNP-dependent cgmp levels in the root stele of Zea mays cgmp (fmol / mg fresh weight) Time (min): control

25 Recombinant AtPNP-A modulates H + fluxes in A.t. roots 2 1 in Average H + fluxes in elongation zone Average H + fluxes in mature region Net H + fluxes in nmoles m -2 s Net flux out Net H + fluxes (nmoles m -2 s -1) Min.: Time (min) Net H + fluxes in nmoles m -2 s ` Time (min) in Net flux out

26 AtPNP-A modulates K + and Na + fluxes in A.t. roots Net K + fluxes in nmoles m -2 s -1 Net K + fluxes in nmoles m -2 s -1 Average K + flux in the elongation zone in Net flux out Time (min) Average K + flux in the mature zone in Net flux out Time (min) Net Na + fluxes in nmoles m -2 s -1 Net Na + fluxes in nmoles m -2 s Time (min) Average Na + flux in the elongation zone Average Na + flux in the mature zone Average Na flux Time (min)

27 Towards a key discovery

28 Monitoring osmotically induced volume changes in mesophyll cell protoplasts Absorbance ( nm) Time (min) 0.2 M 0.4 M 0.6 M MCP diameter in µm Sorbitol conc. (M) 0.2 M 0.4 M 0.6 M Sorbitol 40 µm

29 AtPNP-A modulates osmoticum-dependent volume changes in MCPs Mean Protoplast Volume (µm 3 ) AtPNP-A: cyclohex. : (µg/ml) AtPNP-A drives H 2 O uptake even under osmotically unfavourable conditions

30 All AtPNP-A dependent processes discovered to date are relevant to plant homeostasis and growth

31 PNP functions - known to date: H + Plasmalemma ATP ADP + P i K + Na + K +, Na + K + K IR PNP PNP H + Cl - ΔH + PNP PNP GTP cgmp PNP PNP guanylate cyclase domain Comp. Sol. Synth. H 2 O low affinity cation transporter

32 In search of a biological role for PNPs WT KO Comp Control 50 mm NaCl

33 Insights from whole plant physiology

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35 Temperature dependence of systemic is indicative for phloem transport

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37 Meier, S., Bastian, R., Donaldson, L., Murray, S., Bajic, V. and Gehring, C. Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides. BMC Plant Biol. (2008) 8, 24

38 Meier, S. and Gehring, C. A guide to the integrated application of on-line data mining tools for the inference of gene functions at the systems level. Biotech. J. (2008) DOI: /biot

39 A surprising finding

40 A bacterium with a plant-like peptide A.thaliana X.axonopodis A.thaliana X.axonopodis A.thaliana X.axonopodis --MAVKFVVVM----IVFAQILAPIAEAAQGKAVYYDPPYTR-----SAC MGIVMKHKILLGFSVAAIGLLFSSAAFADIGTISFYGNNARRPADLVQGC :.:*. :::.:. :::. * * *. :*. *..* YG-----TQRETLVVGVKNNLWQNGRACGRRYRVRCIGATYNFDRACTGR NVPEDQVSGRNYQVVTVSDGLWDNGASCGRRYRMRCISTPVKHS--CTAS : *: ** *.:.**:** :******:***.:. :.. **. TVDVKVVDFCREPCNGDLNLSRDAFRVIANTDAGNIRVVYTP---- TIDVIVVGRCPN-GRCTVGGRDVTMKIAFNRYSLLVQARTAPWANI *:** **. * :. :. :::: * : ::. :*

41 A HYPOTHESIS: Molecular mimicry of a signalling molecule may permit pathogen control of plant homeostasis

42 XacPNP works in plants

43 XacPNP induction by apoplast-like conditions

44 XacPNP weakens host defense

45 Out-look: Structural and functional characterisation of recombinant molecules Search of the PNP receptor Applications of PNPs in rational approaches to increasing abiotic stress tolerance

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