The jaz family of repressors is the missing

The JAZ family of repressors is the missinglink in jasmonate signalling

A. Chini, S. Fonseca, G. Ferna´ndez, B. Adie, J. M. Chico, O. Lorenzo, G. Garcı´a-Casado, I. Lo´pez-Vidriero, F. M. Lozano, M. R. Ponce, J. L. Micol & R. Solano

November 13th , Tuesday, 2007

Kihye Shin

NATURE Vol 448 9 August 2007

Jasmine

Jasmonates

JA and MeJA inhibit the germination of nondormant seeds and stimulate the germina-tion of dormant seeds

High levels of JA encourage the accumulation of storage proteins; genes encoding vegetative storage proteins are JA responsive and tuberonic acid (a JA derivative) has been proposed to play a role in the formation of tubers

JA application can induce chlorosis and inhibition of genes encoding proteins in-volved in photosynthesis, although the purpose of this response is unknown it is proposed that this response to JA could help reduce the plant's capacity for carbon assimilation under conditions of excess light or carbon

The role of JA accumulation in flowers and fruit is unknown; however, it may be re-lated to fruit ripening (via ethylene), fruit carotenoid composition, and expression of genes encoding seed and vegetative storage proteins

JA plays a role in insect and disease resistance. Many genes during plant defense are induced by JA; JA and ethylene may act together in defense response

Jasmonates

key regulators of plant responses to environmental stresses and biotic challenges

ozone exposure Wounding water deficit pathogen and pest attack

involved in important plant developmental processes

root growth Tuberization fruit ripening tendril coiling reproductive development senescence

Jasmonate signaling pathway

JA

COI1(SCF complex)

AtMYC2

DHS1, VSP1, LEC

Reduced sensitivity to JA mutants

Mutant Characteristics (screen/phenotype) Gene

coi1  

Reduced root growth inhibition and anthocyanin accumulation by coronatine (and JA). Male-sterile. Enhanced sensitivity to A. brassicicola, Pythium irregulare and Bradysia impatiens.

F-box leucine-rich re-peat (LRR) (At2 g39940)  

jar1/jin4/jai2 

Reduced root growth inhibition by MeJA. Enhanced sensitivity to Pythium irregu-lare.

JA–amino synthetase (At2 g46370) 

jin1/jai1 

Reduced root growth inhibition by JA. AtMYC2 is a nuclear-localised bHLHzip transcription factor.

AtMYC2 (At1 g32640) 

jai3 Reduced root growth inhibition by JA in ein3 background.  

mpk4 

Alteration in the expression of JA- and SA-response genes. Dwarf phenotype. AtMPK4 (At4 g01370) 

jai4/sgt1b Reduced root growth inhibition by JA in the ein3 background. SGT1b (At4 g11260)

axr1 Reduced root growth inhibition by MeJA.RUB-activating en-zyme E1 (At1 g05180)

jue1–3 Reduced expression of pLOX2::LUC.  

oji1  

Enhanced sensitivity to ozone. Reduced root growth inhibition by MeJA.Reduced expression of VSP.

Identification of jai Mutants

Ethyl methane sulfonate (EMS) mutagenized ein3-3 plantsmutants were insensitive to JA

JASMONATE-INSENSITIVE1 (JAI1/JIN1). JIN1 encodes AtMYC2, a nuclear-localized basic helixloop-helix-leucine zipper transcription factor, whose expression is rapidly upregulated by JA, in a CORONATINE INSENSITIVE1–dependent manner

Thirty-one genes showed a lower expression in the jai3-1 mutant

Table S1: Differentially expressed genes in jai3-1 vs wild-type plants , after JA treatment (FDR<0.1 and a log ratio >0.5/ <-0.5 in at least one experiment). 35S:AtMYC2 values are also included for comparison. A, signal value (in log scale), M, signal log ratio, FDR, false discovery rate p-values

JAI3-dependent gene expression

Meta-analysis of downregulated genes showedthat most of them were upregulated by jasmonate in wild-type plants

wild-type JAI3 is required for a complete response to jasmonate and that the dominant jai3-1 mutation represses jasmonate-dependent gene expression

identification of JAI3

A positional cloning approach

identify At3g17860 as the mutated gene in the jai3-1 mutant

JAZ (jasmonate ZIM-domain proteins)

a member of a previously uncharacterized family of 12 proteins of un-known biochemical function

JAZ (jasmonate ZIM-domain proteins)

Phenogram representation of the neighbour-joining for proteins

JAZ proteins show domain simi-larity with transcription factors,such as ZIM1 and CON-STANS (CO) that may imply a function as transcriptional regu-lators.

none of the JAZs contains any known DNA-binding domain

identification of jai3-1 mutants

Sequencing of At3g17860 genomic DNA in the jai3-1 mutantidentified a G to A transition in the splicing acceptor site of its 5th intron

lacking the C-terminal domain and including 14 extra amino acids en-coded by the fifth intron

dominant jasmonate-insensitive phenotype of jai3-1

the importance of the C-terminal domain in the regulation of JAI3 activity

physical interaction between JAI3and COI1 proteins

enrichment of [35S]JAI3 in amylose resins containing maltose binding protein (MBP)–COI1 compared to MBP controls

COI1 interacts with the JAI3 N terminus (containing the ZIM domain), but not with its C terminus (containing the C-terminal domain)

proteasome degradation of JAI3

JAI3–GFP (green fluorescent protein) in wild-type

disappeared after jasmonate treatmentThe proteasome-specific inhibitor MG132 blocked JAI3 degradation

the 26S proteasome in jasmonate-mediated JAI3 removal

proteasome degradation of JAI3

JAI3–GFP (green fluorescent protein) in coi1-1

JAI3 is a target of SCFCOI1

proteasome degradation of JAI3

the C-terminal domain is required for JAI3 instability in response to jasmonate, degradation of JAI3 is required for jasmonatesensitivity.

JAI3∆C–GFP (green fluorescent protein) in wild-type

Proteasome-mediated, COI1-dependent degradation of JAI3 in vitro.

Prevention of proteasome-mediated degradation of JAI3 by JAI3ΔC inNicotiana benthamiana leaves.

Regulation of MYC2 by JAI3

Regulation of MYC2 by JAI3

MYC2 interacts with MBP–JAI3∆N, containing the C-terminal domain

JAI3 interacts with the N terminus of MYC2

Yeast two-hybrid interactionsbetween JAI3, MYC2

Interaction model

MYC2

SCF COI1

ZIMJAI3

Regulation of MYC2 by JAI3

most of the genes showing a lower expression in jai3-1 than in wild-type plants after jasmonate treatment are regulated by MYC2

JAI3 represents the missing molecular link be-tween the two wellestablished steps of the jas-monate signalling pathway, SCFCOI1 andthe transcription factor regulating jasmonate-de-pendent transcriptional reprogramming

Functional redundancy among JAZ proteins

other JAZ proteins (for example, JAZ1) also interact with COI1 and MYC2, and are similarly degraded by the proteasome

Feed-back regulation of JAZ genes by MYC2

JA–Ile

Figure |S7. Proteasome degradation of JAI3 in vitro requires JAR1 modification of JA.Quantification of [35S]JAI3 residual protein following incubation with cellular extracts fromjar1 mutant plants after indicated treatments and incubation times. JA: 50 μM JA; MG132:200 μM of the proteasome specific inhibitor MG132. Control, - JA. The values plotted are the mean (+ standard deviation) of three indepen-dent experiments

The requirement of JAR1 for JAI3 degra-dation after JA treatment, but not after coronatine treatment, suggests that a JA-derivative (likely JA-Ile) is the active hormone rather than JA itself. Moreover, coronatine may also be an active ana-logue

JAZ (jasmonate ZIM-domain proteins)

Identification and analysis of eight JAZ pro-teins in Arabidopsis

Deletion of domain 3 of JAZ1 produces a dom-inant, jasmonate resistant phenotype

Analysis of JAZ1–GUS function

JA–Ile-dependent interaction between COI1 and JAZ1 in yeast

Interaction between tomato COI1 and JAZ1 in yeast was also de-pendent on the presence of JA–Ile, and was not stimulated by jasmonate, MeJA or OPDA

Specificity of jasmonate action in a cell-free system

COI1 and JAZ1 interact physically, and that the interaction is promoted in a highly specific manner by JA–Ile

summary

a, In the absence of JA, JAI3 interacts through its N-terminal (ZIM-containing) portion with COI1 and through its CT domain with AtMYC2 maintaining this TF inactive. b, Upon JA perception, SCFCOI1 promotes the ubiquitination of JAI3 and its subsequent degradation by the 26S pro-teasome. JAI3 degradation liberates AtMYC2 that subsequently activates transcription of early JA responsive genes (includingJAZs). Expression of JAI3 and other JAZs restores the un-in-duced situation by re-repressing AtMYC2

summary

The mutated JAI3ΔC protein is resistant to proteasome degradation. Continuous binding of JAI3ΔC to SCFCOI1 prevents ubiquitination and degradation of JAI3 and otherJAZs, thus maintaining AtMYC2 inactive

Discussion

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