cGMP and guanylyl cyclases in plant

signaling

The bacterial pathogen Xanthomonas axonopodis uses a plantnatriuretic peptide-like protein to modify host responses - acase of molecular mimicry

The nature of second messengers and in particular cGMP

Novel aspects of gene discovery

SECOND MESSENGERS

  Second messengers transmit information

  Second - because they link environmental stimuli - via hormones - to effector molecules often resulting in changes of transcriptome and proteome

  Second messengers must be mobile, transient and stimulus/response specific

Ca2+ is arguably the best studied second messenger

  How can Ca2+ mediate plant responses to   both auxin and ABA ?

  - Temporal and spatial signatures   - Specific cooperation with other messengers   e.g. [pH]i, cADPR, cAMP, cGMP, IP3

cGMP - GUANOSINE 3’,5’-CYCLIC MONOPHOSPHATE

  GTP ➔ {GC} ➔ cGMP + 2Pi

  The presence of cGMP in plants has been known for many decades

  A serious interest in the role of cGMP as a second messenger in plants is recent (90-ies)

  PS: There is a NY connection

Guanosine is a nucleoside comprising guanine attached to a ribofuranose ring via a β-N9-glycosidic bond.

Guanosine can be phosphorylated to become GMP (guanosine monophosphate), cGMP (cyclic guanosine monophosphate), GDP (guanosine diphosphate) and GTP (guanosine triphosphate).

PS: The history of cAMP & ACs in plants is nothing short of dramatic - See PubMed

cGMP - DEPENDENT PROCESSES

  Are:   Processes that are inhibited in the presence of GC

inhibitors or processes that can be triggered and/or sustained by [8-Br-] cGMP

  E.g.:   - Gating of channels - major biotechnological implications - Phytochrome signalling   - Transcription of specific target genes   - NO-dependent stress signalling

0 5 10 15 20

0.0

0.5

1.0

1.5

2.0

30 90 150

control

Time (min):

cGMP (fmol / mg

fresh weight)

Δ [cGMP] in response to PNP

cGM

P in

fmol

/mg

fres

h w

eigh

t

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

H2O

50 mM NaCl

150 mM NaCl

100 mM Sorbitol

300 mM Sorbitol LY83583

Neomycin

[cGMP] changes differentially in response to NaCl and dehydration stress

cGMP - DEPENDENT PROCESSES

cGMP

sGC pGC

Nucleus

cGMP

cGMP

cGMP

K+, Na+

ATP

ADP + Pi

H+ Cl-

ΔH+

K+ Na+

Cell wall

Ca2+ Ca2+

H20

H+

Where are the GCs in higher plants ?

Why were there no higher plant GCs annotated or published in 2002?

-  Blast searches with annotated GC from lower and higher eukaryotes return no GCs in higher plants

-  The major reason could be gene loss and subsequent gain

Our hypothesis: Only the catalytic centre is conserved in higher plant GCs

The search strategy: Bio-informatics, structure modelling, biochemistry and cell biology

Defining the catalytic centre of GCs:

1.  Residues that do the hydrogen bonding with the guanine

2. Residues that confer substrate specificity (AC/GC) 3. Residues that stabilise the transition interphase 4. Residues that do the metal ion binding (Mg2+ or Mn2+)

ALIGNMENT OF GC CATALYTIC CENTRES

E: Glutamic acid *

Catalytic region of class III NC as a reference:

From: McCue et al. Genome Res. (2000) 10: 204-219. The Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509 USA

E: Glutamic acid *

T.rubripes GSVLAGVVGVKM-PRYCLFGNNVTLANKFESCSQPGKINIS - I

D.melanogaster GEVVTGVIGNRV-PRYCLFGNTVNLTSRTETTGVPGRINVS – II

C.elegans GPCVAGVVGKTM-PRYTLFGDTVNTASRMESNGEALRIHCS - III

H.sapiens GPCVAGVVGLTM-PRYCLFGDTVNTASRMESTGLPYRIHVN - IV

X.laevis GPVCAGVVGLKM-PRYCLFGDTVNTASRMESNGEALKIHVS - V

C.reinhardtii GPATSGVVGQKM-PRFCLFGDTVNTASRMESTGRPGCIHIS - VI

S.dubia GPVVSSVVGQMN-RRYCLFGDSVNTASRMESESKPNRIQVS - VII

Synechocystis GEVVVGNIGSEKRTKYGVVGAQVNLTYRIESYTTGGQIFIS - VIII

D.discoideum GPVVGGIIGKKKL-SWHLFGDTINTSSRMASHSSIGRIQVS - IX

1 7 14 16

THE ORIGINAL SEARCH MOTIF

[RKS][YFW][CTGH][VIL][FV]G[DNA]X[VIL] X{4}[KR] 1 2 3 4 5 6 7 8 9 10-13 14

Motif search: 7 candidate molecules - with a replacement in position “6”

➔

THE FIRST PREDICTED CANDIDATE

[RKS][YFW][CTGH][VIL][FV]G[DNA]X[VIL] X{4}[KR] 1 2 3 4 5 6 7 8 9 10-13 14

AtGC1: G-ILDGN-GDSTFPKYCLFDDPLVSDGKYRDAGLPSSSHMD 1------------14

Metal binding residue/position; E: Glutamic acid

D: Aspartic acid *

*

AtGC1:

M

14.3

20.1

30

45

66 kDa:

➔

(A)

0

1

5

10

15

20

cGM

P (fm

ol/

g pr

otei

n)

25 (B)

- + Mn2+:

- +

Mg2+: + + - -

- + - + - +

+ - a b c d e f g h

AtGC1 has GC activity in vitro

- AtGC1 can function as monomer or homo oliogomer - AtGC1 soluble but not NO sensitive - AtGC1 is a novel type of GC - To-date, no NO binding molecule is known in plants

From: Ludidi and Gehring, J.Biol. Chem. 278, 6490-6494, 2003

We are currently elucidating the biological role of AtGC1

Ginalski K, Zemojtel T. ECEPE proteins: A novel family of eukaryotic cysteine proteinases. Trends Biochem Sci 2004; 29:524 - 6.

DOMAIN ORGANISATION OF KNOWN GCs

PS: Most soluble GCs known to-date have heme-binding Domains and are NO-responsive

We have good reasons to believe that AtGC1 is not the only GC in higher plants

(1)   Chlamydomonas has > 90 annotated NC

(2)   Chlamydomonas GCs come in > 20 domain combinations

(3) NO donors trigger cGMP transients in higher plants

(4) Membrane preparations may have GC activity

THE SEARCH FOR ADDITIONAL PLANT GCs

DOMAIN ARCHITECTURE OF AtGC-WAK-L

-  Similarity to ANP-receptor - Is AtGC-WAKL a PNP receptor?

THE CYTOSOLIC

DOMAIN HAS GC

ACTIVITY IN VITRO

Is AtGC-WAK-L a receptor ?

The AtGC-WAK-L response to Pseudomonas

0

500

1000

1500

2000

2500

3000

3500

4000 Si

gnal

inte

nsity

Hours: 0 6 2 24 6 2 24 6 2 24

VIRULENCE C AVIRULENCE MgCl2

4500

5000

PS: The avirulence response leads to a more pronounced cGMP increase than the virulence response

Further evidence for receptor type GCs: The Brassinosteroid receptor

From: Kwezi et al., PLoS one , 2007

Note: Motif extensions are based on rational considerations and site-directed mutagenesis.

D: Aspartic acid

NO RIDDLE

Known soluble GCs signal via NO

Soluble plant GCs do not seem to have NO (heme-) binding domains

NO stimulates cGMP synthesis in planta

Apoptosis

Avirulence signal/receptor

cGMP

PR proteins

cADPR

Flavonoids & Phytoalexins

SA

H2O2

From: Delledonne, M. (2005) Current Opinion in Plant Biology, 8, 390-396.

IN SEARCH OF NO-BINDING PLANT MOLECULES

Nature Chemical Biology June 2005

IN SEARCH OF NO-BINDING PLANT MOLECULES

Extracted H-NOX motif: Hx{12}Px{14,16}YxSxR

The 4 A.t. molecules with an H-NOX motif:

IN SEARCH OF NO-BINDING MOLECULES

H-NOX motif: Hx{12}Px{14,16}YxSxR Relaxed catalytic GC motif: [RKS]x[GHCTS]x{6}[NT]x{3}[KR]

Mainly sol. GCs and NBS-LRR with a role in disease resistance Populus trichocarpa (Western balsam poplar)

IN SEARCH OF NO-BINDING PLANT MOLECULES

H-NOX motif: Hx{12}Px{14,16}YxSxR

-MVPAVNPPTTSNHVAVIGAGAAGLVAARELRREGHSVVVFERGNHIGGV WAYTPNVEPDPLSIDPTRPVIHSSLYSSLRTIIPQECMGFTDFPFSTRLE NGSRDPRRHPGHSEVLAYLRDFVREFKIEEMIRFETEVVRVEQAGENPKK WRVKSRNFGDISDEIYDAVVVCNGHYTEPRHALIPGNKINHSFSIGLGID TWPGKQIHSHNYRVPEQVKDQVVVVIGSSVSGVDISRDIANVTKEVHISS RSTKPETYEKLPGYDNLWLHSNIETVREDGSVVFKNGKTVYADTIMHCTG YKYYFPFLDTKGEVTVEDNRVGPLYKHVFPPALSPGLSFIGLPWQNMKLQ TLDVNELIGQCFGYLLVIPFPMFELQSKWVAAVLAGRVSLPSQEEMEDTK MFYLKLEASCIPKRYTHLMAELDSQFVYNNWLADQCDYPRIEKWREQMFY KVFKRIQSQASTYKDDWDDDHLIAEAYEDFVKFPSNYPSSLIEREYTS

Annotated as mono-oxigenase; N-oxide forming homologues

MAVLRLHSSSSTSSSSPSSFSPHTERQTKKKRSRNXGLFFRADFSFRLELGFLIRRVRVL GEIFCLFVCFSDGESIDLEWETGGLRVCIGGGDWGIRGPLYGLNSILMCNKGIARVSDSV DQKQHQAVYLMDSPSATPSSAXGSNDENPRVKFLCSFSGSILPRPQDGKLRYVGGETRIV SVPRDIGYEELMGKMKELFDMAAVLKYQQPDEDLDALVSVVNDDDVTNMMEEYDKLGSGD GFTRLRIFLFSHPDQDGGSSHFVDVDDTERRYVDALNNLNDASDFRKQQVGESPTMSAID DIHLAEQFFNSISLEGGLHNQRNCEMPMSQFNLHHLTIPHMGSGQHQPVAQRYNEMESQW NPAYFSPRHHGHHDARPLAEYPSSPSSARFRMPFGELPDKCIDRXPEEYSRQPVNPQAPY DHQPQASDNVVWLPTGAISSEKAGFPGSMLHGPNVFEGNSICEHCRMTFHRHLEQPNMGN GLPPVANPCAECPPGRESFLLNTDAKMQHGIYPKEHNDPRSLYNETHNHERGWILQHQLN PRAEDARAQISGAGRLNDPYIVDGSGVNFPVAHGNLLDNHHVSSNYVHHEDXRYIRTGXE LGNGVFHDQAAAAGPAINVPPLEERAVRYGNLPYPYGADNLYQVSHGHVPAHALWRNVQN PMHGAPSYEASTSTCQAXGSVNPGPIRGTREGSPRFCVGLDNQNPWGESSQKILGFDGSA LPDYSYGHATKLNPNTHGQEGQHPFTPGPVPSPSDMLKFAAPMEPLHFTNSSPTLMDDKF VASANLSYNPESRNDNNVNQTVIMEAKQAFREGKEEIHMEKVEDNDMPVTSLPEKNNNAD KKCEVASLEPVNLPAEDNVFKPVVNDCAPLEEDAKLDVSNLSFLPELIASVKRAALESAE EVKAKVQENADAVHASSTKEASNELETANALGDLELDSDNDNVNTFKIEPTKAEEEALSR GLQTIKNDDLEEIRELGSGTYGAVYHGKWKGSDVAIKRIKASCFAGRPSERERLVIICFA SIEVGLSLVPSVYNIGLVGFIADMKMSFCIIDLVLSDAYIKAWVNGIGWDVGAAGTDIFK EFQRSTSHCLAEKLKSLKRDLRRWNKEVFGNVSAKKVEALSQIXFWDSKACLNPLSSEEA EARLGDLEEYKKCVLMEETFWRQKSRETWLKEGDKNTKFFHKMXNARARKNLLSKVNING NSLTSAEDIKDGGGAKELKDFRPISLVGSFYKLLAKVLANRLKQXIGEVVSEYQHAFIRN RQILDAALIANETVDSRLKVNIPGLLLKLDIEKAFDHVNWDCLVSVMSKMGFGQKWINWI SWCISTTNFSILINGTPSDFFRSTRGLRQGDPLSPYLFLLVMEADSGQLRYLSWVLLWFE AISGLXVNRDKSEVIPVGRVDYLENIVSVLGCRIGNLPSSYLGLPLGAPFKSPRVWDVVE ERFRKCLSLWKRQYLSKGGRLTLIKSTLSSLPIYLMSLFVIPRKVCARIEKIQRDFLWGG GALEKKPHLVNWSAVCTDMRQGGLGIRSLVALNRALLGKWNWKFSIERNSLWKQVIIDKY GEEEGGWCSKEVRGAYGVGLWKAIRKDWEIIRSRSRFIVGNGRKVKFWKDLWCEDQALED AFPNLFRLAVNKNQWVCDAWEEEGEQADENNFVNLGEFNLDALRVLFWPGMMTVKSPVSS PGGKCWFGIDLKSFEINIEDHKGKVRGKICERGPKFSSWIRFGGKGLSLLLEGVESVCGL KERTPFRKFWSEGDRVYXLELRGRGLVGGWKMLASKLRSVGVAPLQWSGVLLDKQSPSQA SPSSSVGRGLCPLRNRPEPRDAVWLEIEKETLDRNEELLGRCLVGSWVGDADRLPDPVSF GSWAKNSWFLEGNLWLSNVRENLMLLEFEFEDEAERVFSSGIRSFRGRSFRLEKWKPSVG CLEGDNEETRHVWVRILGLPLHLWGRSLFKQFGDACGRYVAVDENTAERRNLKWAKVLVE TSGWQHPSSLQVVAGATCYALQLWWEEEPCLSSVIPACRSGAWKIGDDVVAPSRAMGSVD PQTPASVTLQPEKLLSPTLGTVAPTSDLTGEAAPSSPARAMEKGQYPLSQARVEESGQLL DALAHSSLACGRPFGPGLGKAQVPMGSSLKPLDPAPSKALRQESPSCSLRRSGAPSATPS FSRAPQKVASPLLDAPTASRPVEASKTPQMEASSPVESPMHAFELSLPVKARKSTQLEAS

HLVEVPTAVKELPSPAEARNKKGTPALPPISLRGDPSSSSSSFWDTGLERGREPGSRPLS PMARDVEVTPNPLSIVLRDGSTVILSTTPTSDLENNMAKQRDSSDYPPGEEGWSEEELSK LLHFSQVLGMPVEGHEVEILELLSKLKLRTGSNSLRKRRKKKKSCSTRFERELKRLECSV SYKGTSGISKRSGLHDCDKRKLIKGVVRNQKADLVCLLETKVKDVSTQLVNSVGVGRFLN WASVDARGTAGGLLLIWDNRVLENLEVESGGYSISVRFRNCSDGFSWIFSGVYGPVIGSE KEDFWEELGAIRGLWEDPWCIGGDFNAVRYPEERRNAPRLTADMRRFSEVIGELGLRDIP LAGGPFTWIGGLNSQAASRLDRFLISDQWEDHFSAISQSALPRLVSDHSPIILEAGGFSS GKSPFRFENMWLKIEGFKDLVKSWWNGYSVEGFSSHCIAEKLKALKKDLKKWNKEVVGNV SFNRAEALSRLQQWEAKENENALTPEDLEAKNLDLEEYKKWALLEETSWRQKSREIWLRE GDKNTKYFHKMANARARRNFLSKIKVNGVYLSSLAEIKEGVCNAYQTLLSDPGDWRPSIN GLNFKELGEGLASSLEVMFSEEEIFAALSSFCGDKAPGPDGFTMAFWLFCWDVVKPEIIG LFREFYLHGTFQRSLNSTFLLLIPKKEGTEDLKDFRPISLVGSVYKLLAKVLANRLKTVM GEVISDSQHAFVHGRQILDXVLIANEALDSRLKDNIPGLLLKMDIEKAFDHVNWNFLMEV MSKMGFGHRWINWIKWCCSTTSFSILINGSPSGFFRSSRGLRQGDPLSPYLFLLAMEALS QLLSRARNGNFISGFRVGGRGSEGLVVSHLLFADDTLIFCDADADQLQYLSWTFMWFEAI SGLKVNLNKTEAIPVGEDIPMETLAAVLGCKIGSLPTSYLGLPLGAPYKSIRVWDAVEER FRKRLSLWKRQYLSKGGRLTLLKSTLSSLPTYFLSLFVIPKRVCARLEKIQRDFLWGGGA LEKKPHLVSWKVVCADKKKGGLGIRSLATFNKALLGKWLWRFANENEPLWKQIILSKYDL QEGGWCSKDARNWYGVGVWKAIRKGWENFRSHSRFIIGDGTKVKFWKDLWCGNQSLKETF PILFNLSVNKEGWVAEAWEEDEGGXSWGLRFNRHLNDWEVGEVESLLSKLHPLTIRRGVE DMFRWKENKIGTFSVKSFYSSFSRDSKPPFPARTIWTPWVPIRASFFGWEAAWNRLLTTD RLKRIGWSIPNRCFLCKHKEETTDHLLLFCEKARMLWLLIFSLFGVQWVMHSTVKNHLLG WHGSFVGKKRKKAWRAAPLCLMWTIWRERNRRAFDDMERNDQDIKSIFLYTFVNWARIAD FWKEALILSSLHHPNVVSFYGIVRDGPGGSLATVTEFMVNGSLKQFLQKKDRTIDRRKRR IIAMDASFGMEYLHGKNIVHFDLKCENLLVNMRDPHRPVCKIGDLGLSKVKQHTLVSGGV RGTLPWMAPELLSGKTNMIDVYSFGIVMWELLTGDEPYADMHCASIIDDKEGRKRDHDLK WEQGGFSSVASGYFKHLWITLQWLGCQQYSQMVTSLSLEGHCTSLPRVFQVYSVCGRRRD RIWFWEDLWWGDQPLGVQYPRLLIVVTDKNTPISSILGSTRPFSWNFNFCRNLSDSEIED LEGLMRSLDRLHISPSVPDMRSWSLSXXGLFTVKSFFLALSQFSDSPPVFPTKFVWNSQV PFKVKSFVWLVAHKKVNTNDLLQLRRPYKALSPDICKLCMKHGKTVBHLFLHCSLTMGLW HRLFQLXKTDWVPPRSISDMLSINFNGFGSSKRGVVLWQDACIAIMWVVWRERNARIFED KTRNSXNFWDSIRFLVSLWAFCSKVFKGIPLNVLQLDWLAVCNPNGLV

Vitis - A5AQB4

Taking the liberty of accepting and S in position 3 -MVPAVNPPTTSNHVAVIGAGAAGLVAARELRREGHSVVVFERGNHIGGV WAYTPNVEPDPLSIDPTRPVIHSSLYSSLRTIIPQECMGFTDFPFSTRLE NGSRDPRRHPGHSEVLAYLRDFVREFKIEEMIRFETEVVRVEQAGENPKK WRVKSRNFGDISDEIYDAVVVCNGHYTEPRHALIPGNKINHSFSIGLGID TWPGKQIHSHNYRVPEQVKDQVVVVIGSSVSGVDISRDIANVTKEVHISS RSTKPETYEKLPGYDNLWLHSNIETVREDGSVVFKNGKTVYADTIMHCTG YKYYFPFLDTKGEVTVEDNRVGPLYKHVFPPALSPGLSFIGLPWQNMKLQ TLDVNELIGQCFGYLLVIPFPMFELQSKWVAAVLAGRVSLPSQEEMEDTK MFYLKLEASCIPKRYTHLMAELDSQFVYNNWLADQCDYPRIEKWREQMFY KVFKRIQSQASTYKDDWDDDHLIAEAYEDFVKFPSNYPSSLIEREYTS

H-NOX motif: Hx{12}Px{14,16}YxSxR (4) Relaxed GC motif: [RKS]x[GHCTS]x{6}[NT]x{3}[KR] (7339)

[RKS][YFW][GHCTS]x{6}[NT]x{3}[KR] (622) RHALIPGIDTWPGK

- SFSIGLGIDTWPGK -

- SFSIGLGIDTWPGK -

Position 10; T - threonine or N - asparagine

Relaxed/Modified GC motif: [RKS]x[GHCTS]x{6}[NT]x{3}[KR] 1 3 10 14

Molecular interactions:

3

1

10 14

GTP

AtNO-GC: - SFSIGLGIDTWPGK -

An other candidate NO-binding molecule is annotated as glycosyltransferase

In particular a UDP-glycosyltransferase that may be specific for auxin

-  Role of auxin

-  Silencing auxin during stress

-  A direct role for NO ? A hypothesis

➔

Out-look

Determine number and functions of NCs in higher plants? In Chlamydomonas reinhardtii it is > 90

Determine structure & function of novel GCs

Characterise cGMP-dependent expression networks

Apply transgenic GC-mutants to enhance stress response traits

Lessons from Chlamydomonas reinhardtii

Stuart Meier University of the Western Cape & KAUST Ndiko Ludidi UWC & Stellenbosch University Lyndon Mungur University of the Western Cape

Cathal Seoighe National Bioinformatics Network – UCT & NUI Vladimir Bajic SANBI & CBRC KAUST

Helen Irving VCP - Monash University, Melbourne

Katherine Denby University of Cape Town & Warwick University Lara Donaldson University of Cape Town & KAUST

Stefania Pasqualini University of Perugia