UNIVERSIDAD AUTÓNOMA DE MADRID
Programa de Doctorado en Biociencias Moleculares
Functional characterization of MYCs TFs in Marchantia polymorpha
TESIS DOCTORAL María Peñuelas Hortelano
Madrid, 2019
UNIVERSIDAD AUTÓNOMA DE MADRID
Programa de Doctorado en Biociencias Moleculares
Functional characterization of MYCs TFs in Marchantia polymorpha
TESIS DOCTORAL María Peñuelas Hortelano
DIRECTOR
Roberto Solano Tavira
Madrid, 2019
UNIVERSIDAD AUTÓNOMA DE MADRID
Programa de Doctorado en Biociencias Moleculares Facultad de Ciencias
Functional characterization of MYC TFs in Marchantia polymorpha
Dr. Roberto Solano Tavira Dr. Francisco Zafra Gómez
Profesor de Investigación Catedrático Universidad DIRECTOR TUTORMaría Peñuelas Hortelano
Licenciada en Ciencias AmbientalesMaster en Biotecnología DOCTORANDA
Madrid, 2019
transformar el dolor en fuerza para superarse y salir
fortalecido de ellas.
ACKNOWLEDGEMENTS
En primer lugar, me gustaría agradecer a mi director de tesis, Roberto Solano. Gracias por toda la paciencia que has tenido conmigo y ayudarme siempre. Sé que a veces no ha sido fácil.
Gracias a todo el 317, solo tengo buenas palabras para todos y cada uno de vosotros. Os voy a echar de menos a todos! Creo que hemos formado un gran equipo.
Gracias a Selena y Gemma “el pasillo oscuro”. Chicas, ha sido un placer compartir todo este tiempo con vosotras. Siempre dispuestas a una risa o a una lágrima. Selena, incluso “a pesar” de tus expandimientos de poyata, tus locuras y los dichosos golpecitos en la silla cada vez que pasabas por detrás...te he cogido cariño, jejej.
Gracias Gemma por los cafés a media tarde y por hacer de muro de contención de mis rayadas mentales en bucle máximo, tanto de dentro y fuera del laboratorio.
Alberto, conquense, que gran descubrimiento!! Muchas gracias por tus ánimos y tus muestras de apoyo y de cariño, sobre todo en estos últimos tiempos. Eres un pequeño gran hombre, y sé que vas a ser un gran científico.
Sophie, thanks for your support. You are a great comedian, think about it…hehe. “Apechusque” do you know??.
Jose y Andrea, gracias por vuestra disposición a ayudarme siempre, por vuestros ánimos y vuestra sabiduría, por vuestro cariño.
Guillermo, cubano, cuanta alegría das a estos cuerpos serranos.
Gonzalo…viva el vino! (me debes una botella, por cierto) Has sido el último en llegar, pero no por ello menos importante.
Gracias a Isa por toda su ayuda, y por enseñarme el “Marchantia world”, ha sido toooooda una experiencia.
No me puedo olvidar de Andrés, te he hechado de menos desde que te fuiste! Cuantas conversaciones mañaneras tempraneras, siempre sacándome de apuros informáticos y/o burocráticos.
Gloria, que pena que te fuiste justo cuando más te estaba empezando a conocer. Ese “hombro” del bioanalizer donde llorar, con mis RNAs en la mano…como podrían salir bien?!
En todo este tiempo, también ha habido otras personas que han estado presentes.
Maremoto, fuiste la primera en enseñarme.
“Javi labo” así te tengo todavía en el móvil. En mis comienzos en un laboratorio estás tú. Me enseñaste a usar la lupa, y desde ahí, todo fue en ascenso. Siempre has estado para mí y no tendré nunca palabras para describir todo lo que me has ayudado y me has soportado durante todos estos años.
Gracias a mis científicas locas, Laura y Carmen, habéis sido un gran pilar siempre, gracias por las llamadas a tres y nuestras quedadas siempre reponedoras. ¡Lo que la ciencia unió que no lo separe nada! Carmen, siempre has sido mi “botón de emergencia”, cuanto me das!! Como no te gustan las moñerías, ahí lo dejo…
Vane, me has acompañado siempre, desde que nos conocimos en Toledo, pasando por Zaragoza, y Madrid. Aunque no entendías lo que te contaba, ni sabías de lo que te hablaba, siempre has creído en mí. Gracias por ser y estar, I love you.
Gracias Fran, por ayudarme a sacar ese nudo del estómago. Por los
“paseos” al CNB a horas intempestivas, por pasar la semana santa delante del ordenador escribiendo este documento. Por entenderme y darme los ánimos y la tranquilidad que necesito y que me falta.
Gracias compi.
Gracias a todos los que me habéis apoyado y/o os habéis preocupado. Las EMES!, Pachi y Kachenka por echarme de menos
los fines de semana, y a Mati por preguntar por la tesis cada vez que me veía.
A mi coctel team!! Gracias chicos, por hacer más amena la ciencia.
No conseguisteis sacarme de la mahou, y no será por no haberlo intentado…Os habéis convertido en muy buenos amigos.
Guille, no olvidaré tu bote de desechos de regalo (no se si era una indirecta) y las charlas quejándonos de todo (hater!!). Ramón,
¡cuanto te he hechado de menos! Desde que te fuiste me ha faltado una parte en el CNB, no es lo mismo. Las visitas al 316 siempre venían bien. Ana, tu también! Coctel team forever!!!
Por supuesto gracias a mi familia, a mi padre, a mi madre y a mi hermano. Gracias por intentar entenderme, aunque a veces fuera difícil, por apoyarme, preocuparos, y querer lo mejor para mi siempre. ¡Os quiero!
TABLE OF CONTENTS
Content
SUMMARY ... 1
ABBREVIATIONS ... 7
INTRODUCTION ... 13
JA biosynthesis ... 13
JA perception and signalling ... 15
Transcription factors. The bHLH Family ... 17
MYC Transcription factors ... 19
Evolution ... 23
Marchantia polymorpha ... 25
OBJECTIVES ... 33
MATERIALS AND METHODS ... 37
Plant material and growth conditions ... 37
Chemicals ... 38
Plant transformation ... 38
Gene identification and phylogenetic analyses ... 38
CRISPR/Cas9 mediated mutagenesis to obtain Mpmycy and Mpmycx mutants ... 39
Fertility assays ... 39
Microarray analysis... 40
Herbivory assays ... 41
Terpene extraction and quantification ... 41
Cloning ... 42
Protein expression, purification and determination of DNA
binding motifs ... 43
Pull-down assays ... 43
Confocal microscopy ... 44
Western blot analysis ... 44
Light Treatments... 45
Yeast two-hybrid assays ... 45
qPCR analysis ... 45
Statistical analysis ... 46
Experimental contributions ... 46
RESULTS ... 49
MYC proteins predate plant terrestrialization ... 49
Nuclear localization of MpMYCs and interaction with MpJAZ .. 51
Constitutive expression of MpMYCs confers hypersensitivity to OPDA ... 55
MpMYCs are required for growth inhibition by OPDA ... 59
MpMYCs are required for OPDA- and wound-dependent gene expression in Marchantia ... 65
MpMYCs regulate sesquiterpene accumulation and defense to herbivory ... 68
MpMYCs are not involved in fertility in M. polymorpha... 70
Conserved regulation of MYC stability by light ... 72
Lack of interspecies MYC complementation ... 73
DISCUSSION ... 81
CONCLUSIONS ... 89
CONCLUSIONES ... 91 BIBLIOGRAPHY ... 95 SUPPLEMENTARY DATA ... 113
SUMMARY
SUMMARY
The lipid-derived phytohormone jasmonoyl-isoleucine (JA-Ile) regulates plant immunity, growth and development in vascular plants by activating genome-wide transcriptional reprogramming.
In Arabidopsis, this is largely orchestrated by the master regulator MYC2 and related transcription factors (TFs). However, the TFs activating this pathway in basal plant lineages are currently unknown. In this thesis, we report the functional conservation of MYC-related TFs between the eudicot Arabidopsis thaliana and the liverwort Marchantia polymorpha, a plant belonging to one of the most basal land-plants lineages. Phylogenetic analysis suggests that MYC function first appeared in charophycean algae, and therefore predates the evolutionary appearance of any other jasmonate pathway component. Marchantia possesses two functionally interchangeable MYC genes, one in females and one in males. Similar to AtMYC2, MpMYCs showed nuclear localization, interaction with JAZ-repressors, and regulation by light. Phenotypic and molecular characterization of loss- or gain-of-function mutants demonstrated that MpMYCs are necessary and sufficient for the activation of the pathway in Marchantia, but unlike their Arabidopsis orthologs, do not regulate fertility. The results show that despite 450 million years of independent evolution, MYCs are functionally conserved between bryophytes and eudicots. Genetic conservation in one of the most basal lineages suggests that MYC function existed in the common ancestor of land plants and evolved from a pre-existing MYC function in charophycean algae.
Jasmonoyl-isoleucina (JA-Ile) es una fitohormona lipídica implicada en la regulación de procesos como defensa, crecimiento y desarrollo en plantas vasculares mediante la activación de respuestas transcripcionales. En Arabidopsis, estos procesos están controlados por el factor de transcripción MYC2 y otros factores de la familia MYC. Sin embargo, los factores de transcripción que activan la ruta del Jasmónico en los linajes de plantas más primitivos todavía no se conocen. En este trabajo, hemos llevado a cabo el estudio de la conservación funcional de los factores de transcripción MYC entre la eucotiledonea Arabidopsis thaliana y la hepática Marchantia polymorpha, que pertenece al grupo más primitivo de plantas terrestres. Análisis filogenéticos sugieren que la función MYC apareció inicialmente en algas carofitas precediendo la aparición en la evolución de cualquier otro componente de la ruta del Jasmónico.
Marchantia posee dos genes MYC, funcionalmente intercambiables entre ellos, uno en machos y otro en hembras. Al igual que AtMYC2 en Arabidopsis, MpMYCs tienen localización nuclear, interaccionan con los represores JAZ y están regulados por luz. La caracterización fenotípica y molecular de mutantes de perdida de función demuestra que MpMYCs son necesarios y además suficientes para la activación de la ruta en Marchantia, pero a diferencia de sus ortólogos en Arabidopsis, no regulan fertilidad. En conjunto, los resultados muestran que, a pesar de 450 millones de años de evolución independiente, los factores de transcripción MYCs están funcionalmente conservados entre briofitos y eudicotiledoneas. La conservación genética en uno de los linajes más primitivos de plantas terrestres sugiere que la función MYC existía en el ancestro común a todas las plantas terrestres y evolucionó de una función MYC preexistente en algas carofíceas.
ABBREVIATIONS
ABBREVIATIONS
10,11-EHT 10,11(S)-epoxy-hexadecatrienoic acid 11-HPHT 11(S)-hydroperoxy-hexadecatrienoic acid 12,13-EOT 12,13(S)-epoxy-octadecatrienoic acid 13-HPOT 13(S)-hydroperoxy-octadecatrienoic acid 16:3 Hexadecatrienoic acid
18:3 α-Linolenic acid
4,5-ddh-JA 4,5-didehydro-jasmonic acid
ABA Abscisic acid
ANAC Abscisic acid responsive NAC ANOVA Analysis of variance
AOC Allene Oxide Cyclase AOS Allene Oxide Synthase ARF Auxin response factor At Arabidopsis thaliana bHLH Basic helix loop helix CaMV Cauliflower Mosaic Virus
cDNA Complementary DNA
COI1 Coronatine-insensitive 1
Col Columbia
COP1 Constitutively Photomorphogenic 1
COR Coronatine
CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease 9
CTS Comatose
d days
dn-OPDA 2,3-dinor-OPDA DNA Deoxyribonucleic acid EGL3 Enhancer of Glabra 3 ERF Ethilene response factor
FDR False Discovery Rate
FR Far Red light
GA Gibberellins
GB5 Gamborg´s B5
GL3 Glabra 3
GFP Green Fluorescent Protein
GID1 GIBBERELLIN INSENSITIVE DWARF1
GO Gene Ontology
gDNA Genomic DNA
HR Homologous recombination HY5 ELONGATED HYPOCOTYL 5
JA Jasmonic acid
JA-Ile Jasmonoyl-L-isoleucine JAs Jasmonates
JAM JA-Associated MYC2-like JAR1 Jasmonate Resistant JAT Jasmonate Transporter
JAZ JASMONATE ZIM-DOMAIN proteins JGI Joint Genome Institute
JID JAZ Interaction Domain
LOX Lipoxygenase
Mapoly Marchantia polymorpha
Mb Megabase
MBP Maltose Binding Protein
MED25 MEDIATOR 25
Mp Marchantia polymorpha
MS Murashige and Skoog
NINJA NOVEL INTERACTOR OF JAZ
OPC-4 3-oxo-2-(2-pentenyl)-cyclopentane-1-butyric acid OPC-6 3-oxo-2-(2-pentenyl)-cyclopentane-1-hexanoic acid OPC-8 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic aci
OPR OPDA reductase
OPDA 12-oxo-phytodienoic acid PCR Polymerase chain reaction
PIF PHYTOCHROME-INTERACTING-FACTOR Pto DC3000 Pseudomonas syringae pv. tomato DC3000 qPCR Quantitative Real-Time PCR
RNA Ribonucleic acid
RT-PCR Reverse transcription PCR SA Salicylic acid
SCFCOI1 Skp1-Cul1-F-box protein Coronatine-Insensitive1
SD Standard deviation
sgRNA Short guide RNA TAD Transactivation Domain TFs Transcription factors
TPL TOPLESS
TPR TOPLESS-related
TT8 Transparent Testa 8
WL White light
WT Wild type
INTRODUCTION
INTRODUCTION JA BIOSYNTESIS
Plants use chemical signals for adaptation to a changing environment and thus control biotic and abiotic stress responses (Wasternack, 2007). The integration of exogenous and endogenous developmental programs depends on complex signaling networks responsible for the activation of adaptive responses. Among these signals, Jasmonates (JAs) are oxylipin phytohormones that play essential roles in plant growth, development and stress responses through activation of a genome-wide reprogramming of gene expression. They also regulate growth-defense trade-offs in response to resource limitations (Chini et al., 2016; Howe et al., 2018; Wasternack and Feussner, 2018). The biosynthesis of jasmonic acid (JA) has been mainly studied in angiosperms, such as Arabidopsis thaliana and tomato. It starts with the release of the
fatty acid precursors linolenic (18:3) and hexadecatrienoic (16:3) acids from the chloroplast membrane (Figure1). Then, by the action of 13-lipoxygenases (13-LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC), linolenic acid is transformed into cis-(+)- 12-oxo-phyto-dienoic acid (OPDA). Similar conversion occurs from hexadecatrienoic acid to form dinor-OPDA (dn-OPDA) (Wasternack and Hause, 2018). Subsequently, OPDA is transported into the peroxisome by the action of the ATP-binding cassette transporter (COMATOSE, CTS; Theodoulou et al., 2005) and reduced to 8-(3- oxo-2-(pent-2-enyl) cyclo-pentyl) octanoic acid (OPC-8) by OPDA reductase 3 (OPR3; Chini et al., 2018; Wasternack and Hause, 2018). In the peroxisome, OPC-8 undergoes three cycles of - oxidation and is converted into 6-(3-oxo- 2-(pent-2-enyl) cyclopentyl) hexanoic acid (OPC-6), 4-(3-oxo-2- (pent-2- enyl)cyclopentyl) butanoic acid (OPC-4), and finally produce jasmonic acid (JA). dn-OPDA follows a similar pathway and is reduced to OPC-6 in the peroxisome, entering the -oxidation pathway to produce JA. JA is transported into the cytosol where it is conjugated with isoleucine by the JA-Ile conjugate synthetase JASMONATE RESISTANT1 (JAR1; Staswick, 2004) to generate the bioactive jasmonate in angiosperms JA-Ile ((+)–7-iso-JA-Ile), which is in equilibrium with its inactive epimer (-)-JA-Ile, Figure 1 (Chini et al., 2018; Wasternack and Hause, 2018). Then, JA-Ile is translocated into the nucleus by the JA/JA-Ile transporters JATs (Li et al., 2017) where it binds to its receptor COI1 (Staswick, 2004;
Thines et al., 2007; Katsir et al., 2008; Fonseca et al., 2009a, 2009b;
Sheard et al., 2010).
Recently, an alternative OPR3-independent pathway for JA biosynthesis has been reported, in which peroxisomal -oxidation of OPDA (without previous reduction by OPR3) produces dn-OPDA,
tetranor-OPDA, 4,5-ddh-JA, and this molecule is then reduced to JA by OPR1 and mainly OPR2 (Chini et al., 2018) (Figure 1).
Figure 1: Biosynthetic pathway of JA-Ile. Jasmonates biosynthesis initiates in the chloroplast (green) and continues in the peroxisome (orange) and cytoplasm (yellow). Then, JA-Ile is translocated into the nucleus (brown) where its binds the receptor. Coronatine (COR) the JA-Ile mimic synthesized by Pseudomonas syringae is also perceived in the nucleus by the COI1/JAZ complex. Alternative OPR3 independent pathway is shown in red (Modified from Wasternack and Song, 2017 and Monte, 2017).
JA PERCEPTION AND SIGNALLING
The JA-Ile perception and signaling cascade is well established in eudicots. In Arabidopsis, the core signal transduction chain linking JA synthesis to hormone-induced changes in gene expression comprised different components: JA-Ile, the SCF-type E3 ubiquitin ligase SCFCOI1, JAsmonate ZIM-domain (JAZ) repressor proteins, the ubiquitin/26S proteasome pathway and several transcription
factors (TFs) that regulates the expression of JA responsive genes (Fonseca et al., 2009a; Chini et al., 2016). The bioactive form of the hormone, (+)-7-iso-JA-Ile, is the ligand of a co-receptor complex formed by the F-box protein CORONATINE INSENSITIVE 1 (COI1) and the JASMONATE-ZIM DOMAIN protein (JAZ) (Chini et al., 2007;
Thines et al., 2007; Katsir et al., 2008; Fonseca et al., 2009a, 2009b;
Sheard et al., 2010; Chini et al., 2016). COI1 is the substrate adaptor of the SCF-type E3-ubiquitin ligase and the receptor of JA- Ile. The hormone-triggered interaction of COI1 with its JAZ co- receptors leads to the ubiquitination and subsequent proteasomal degradation of the JAZs (Chini et al., 2007; Thines et al., 2007). In the absence of the hormone, JAZs are repressors of transcription factors (TFs) from different families, including the basic Helix-Loop- Helix (bHLH) TF MYC2, which was the first identified JAZ target (Lorenzo et al., 2004; Chini et al., 2007, 2016). JAZs repress TF activity by preventing interaction with MED25 and by recruiting the co-repressor TOPLESS (TPL) and TPL-related (TPR) proteins through the adaptor protein NINJA (NOVEL INTERACTOR OF JAZ;
Pauwels et al., 2010; Howe et al., 2018, Figure 2). In the presence of JA-Ile, degradation of JAZ repressors leads to a transcriptional reprograming, which includes expression of JAZ genes. Thus, the repression and activation mechanism of the JA-Ile signaling pathway is regulated by a feedback loop, in which JA-Ile responsive genes and their hormone-dependent expression restores the basal situation. Coronatine (COR) is the only known natural ligand of the COI1-JAZ complex besides JA-Ile. This phytotoxin produced by the pathogen Pseudomonas syringae, mimics JA-Ile action and is even more active than the natural hormone (Fonseca et al., 2009a; Yan et al., 2009; Sheard et al., 2010).
Figure 2: JA-Ile signaling pathway. (Model from Monte, 2017). In basal conditions, JAZ proteins repress MYC TFs and recruit a co-repressor complex formed by TOPLESS and NINJA adaptor. The JA-Ile receptor is formed by COI1 and a JAZ co-receptor. JA-Ile perception by the co- receptor complex triggers JAZ proteins ubiquitination and degradation by the 26S proteasome. Elimination of JAZs releases MYC TFs that now can interact with the MEDIATOR complex through the MED25 subunit, which connects the TFs with the general machinery of transcription to activate JA responses. Domains of MYC, JAZ and NINJA are shown in the figure.
TRANSCRIPTION FACTORS. The bHLH FAMILY
Transcription factors (TFs) are master regulators controlling gene expression during development and responses against external stimuli in plants, through their interaction with cis-regulatory elements in their target genes (Ng et al., 2018; Sun et al., 2018). In higher plants, there are about 58 families of TFs regulating the signaling cascades triggered by the perception of a particular hormone. Among them, bHLH (basic helix-loop-helix) are major regulators of jasmonate responses (Goossens et al., 2016).
The bHLH superfamily was first discovered in murine muscle development studies (Sun et al., 2018) and comprises a large number of proteins found in all eukaryotes including fungi, animals and plants. In Arabidopsis, this proteins are involved in processes as light signaling, brassinosteroid and abscisic acid signaling, gynoecium development, abiotic stress responses, flavonoids
biosynthesis, flowering time control, axillary meristem formation, trichome and root hair differentiation, and stomatal patterning (Skinner et al., 2010; Zhao et al., 2012).
Four subclades of the bHLH TF family are implicated in JA signalling in Arabidopsis with different contribution to JA responses: subclade IIIe, IIId, IIIf and IIIb. Subclade IIIe is known as the “positive” JA subclade as it is mainly formed by MYC TFs. MYC2 is seen as the central regulator of JA signaling because it is involved in almost all JA related responses (Kazan and Manners, 2013). The IIId subclade is constituted by the JA-associated MYC2-like (JAM) TFs that negatively regulate JA responses. Members belonging to the IIIf subclade, are involved in particular JA responses such as anthocyanin production and trichome development. This subclade includes TRANSPARENT TESTA 8 (TT8), GLABRA3 (GL3) and ENHANCER OF GL3 (EGL3), which acts through the WD-repeat protein TRANSPARENT TESTA GLABRA 1 (TTG1) and an R2-R3 MYB TFs to form trimeric WD-repeat-bHLH-MYB complexes. MYC1 is also involved in these complexes but direct link to JA signaling has not been probed. The IIIb subclade involved in JA signaling are mainly represented by INDUCER OF CBF EXPRESSION1 (ICE1) and ICE2, that are regulators of freezing tolerance (Goossens et al., 2016).
Among land plants, the genomes of model angiosperm species such as Oryza sativa and Arabidopsis thaliana contain over 150 bHLHs (Pires and Dolan, 2010). This family is also abundant in lycophytes such as Selaginella moellendorffii with 103 bHLH-encoding genes, mosses as Physcomitrella patens with 98 genes and liverworts as Marchantia polymorpha with 51 genes (Bowman et al., 2017). In contrast, only a small number of bHLHs are found in chlorophytes genomes, such as Chlamydomonas reinhardtii and Ostreococcus
tauri encoding three and one bHLH respectively, or red algae such as Cyanidioschyzon merolae, which has only one bHLH (Pires and Dolan, 2010). Therefore, the diversification of plant bHLH proteins was established after the appearance of the first land plants over 450 million years ago (Pires and Dolan, 2010). However, when the MYC subclade appeared within the bHLH family has not been addressed so far.
MYC TRANSCRIPTION FACTORS
Subclade IIIe of bHLH TFs is represented by MYC2, MYC3, MYC4 and MYC5. MYC2 was first identified in Arabidopsis as JASMONATE-INSENSITIVE1 (JAI1/JIN1), that is rapidly upregulated by JA in a COI1-dependendent manner (Lorenzo et al., 2004). MYC2 regulates different responses to JA, acting redundantly with MYC3 and MYC4 to activate defense gene expression, stomatal closure, and accumulation of glucosinolates, terpenoids, and other secondary metabolites (Hong et al., 2012;
Kazan and Manners, 2013; Schweizer et al., 2013; Gimenez-Ibanez et al., 2017). MYC2 acts as transcriptional activator and repressor, positively regulating the expression of early wound-responsive genes (as VSP1), and negatively regulating the expression of late pathogen-responsive genes (PDF1.2, CHIB; Cheng et al., 2011; Zhai et al., 2013). MYC2, MYC3 and MYC4 are nuclear proteins, targets of JAZ repressors, and the interaction JAZ-MYC is also necessary for the nuclear import of JAZs (Chini et al., 2007; Fernández-Calvo et al., 2011; Withers et al., 2012). MYC2 is mainly expressed in roots while MYC3 and MYC4 show stronger expression in aerial parts of the plant. This differential expression patterns links with the control of different JA responses: whereas MYC2 mainly (but not exclusively) control root responses, MYC3 and MYC4 are more important in regulation of responses in the aerial part (Cheng et al.,
2011; Fernández-Calvo et al., 2011). MYC5 regulates stamen development and pollen viability (Figueroa and Browse, 2015), and recently it has also been involved in JA regulated root growth inhibition, plant defenses against insect and pathogens and the positive regulation of JA-induced leaf senescence (Song et al., 2017).
For the regulation of these subset of responses, MYC2, MYC3 and MYC4 bind to the G-box (5´-CACGTG-3´) and G-box related hexamers (CATGTG and AACGTG) in the promoters of their target genes with almost the same DNA binding specificity (Fernández- Calvo et al., 2011; Godoy et al., 2011; Schweizer et al., 2013) . Among MYCs targets, JAZ genes are transcribed in response to JA- Ile to shut down the activation of the pathway through a negative feedback loop (Figure 2).
In addition to the positive MYCs regulators of JA responses (MYC2, 3, 4 and 5), the closely related subclade IIId of bHLH (associated MYC2-like (JAMs) or bHLH003, bHLH013, bHLH014, bHLH017) acts redundantly to negatively regulate JA responses. Similarly, to MYCs, JAMs form homo and heterodimers, and interacts with JAZ repressors. They are also nuclear proteins, and bind DNA with similar binding affinity than MYCs, but they lack the TAD domain, which suggested that their repression activity is achieved by competition for the same cis-regulatory elements (Song et al., 2013;
Fonseca et al., 2014).
MYCs coordinate JA responses with other phytohormones, such as salicylic acid (SA), abscisic acid (ABA), gibberellins (GAs) and auxin (Chini et al., 2016). MYC2 regulates Pst DC3000-mediated suppression of the SA pathway by activating the expression of NAC TFs ANAC019, ANAC055, and ANAC072, which repress the expression of genes involved in SA biosynthesis and metabolism.
The antagonistic interactions between SA and JA pathways increase
resistance to biotrophs by enhancing susceptibility to necrotrophs and viceversa (Gimenez-Ibanez et al., 2017). ABA activates MYC2 expression in a COI1 dependent manner, suggesting that ABA precedes JA in the activation of MYC2-mediated wound responses (Lorenzo et al., 2004; Lorenzo and Solano, 2005). MYC2 positively regulates the MYC2-ANAC019-ANAC055 branch of the JA pathway leading to the expression of the insect defense gene VSP1, whereas negatively regulates the ERF1-ORA59- branch leading to the expression of the pathogen defense gene PDF1.2 (Kazan and Manners, 2013). The crosstalk between JA and GA signaling pathways regulates the balance between growth and defense, and involves DELLA proteins (repressors of GA pathway), JAZ proteins and MYC2. At low GA levels, DELLAs interact with JAZ and allows MYC2 to modulate JA-response genes. Moreover, MYC2 activates the expression of DELLA protein RGL3 that interacts with JAZ repressors and produces the activation of JA-responsive genes in a positive feedback loop. In addition, MYC2 also positively modulates JA and GA related expression of the sesquiterpene synthase biosynthesis genes TPS11 and TPS12 (Kazan and Manners, 2013).
Besides the crosstalk with other hormones, MYCs also link JA signaling to other pathways such as light and the circadian clock, and regulate developmental processes including lateral root formation, flowering time, fertility and SAS (Shade-Avoidance Syndrome; Wasternack and Hause, 2013; Chico et al., 2014; Qi et al., 2015a). Recent studies have identified JA to repress growth in darkness by MYC2-dependent inhibition of COP1 activity (Zheng et al., 2017). In Arabidopsis, MYC2, MYC3 and MYC4 are short-lived proteins degraded under dark or shade conditions, whereas light and JA stabilize them. In contrast, shade stabilizes JAZ repressors and reduces their degradation by JA. Moreover, phytochrome B
(phyB), is required for MYCs stability, since phyB inactivation by FR reduces MYCs stability and JA-mediated plant defenses, suggesting that destabilization of MYCs in shade could be due to phyB inactivation (Chico et al., 2014). Recent results in our lab (Ortigosa, 2018) have identified MYCs as photomorphogenic regulators and (indirect) targets of PhyB. Thus, MYCs are required for modulation of activity of other photomorphogenic factors such as HY5 (ELONGATED HYPOCOTYL 5), explaining the growth/defence trade-off at the molecular level.
Structurally, MYCs possess a bHLH domain in their C-terminus that consists of a basic region of 10-15 amino acids, which are necessary for DNA binding, and another 40-amino-acids segment that forms two amphipathic -helices separated by a loop of variable length (HLH). This HLH domain is responsible for the formation of homo- or heterodimers between bHLH proteins (Kazan and Manners, 2013) (Figure 3).
Figure 3: MYC structural domains. JID, JAZ Interaction Domain;
TAD; TransActivation Domain (also required for JAZ interaction together with JID and for interaction with MED25) bHLH and ZIP domains required for DNA binding and dimerization.
In their N-terminus, MYCs contain a transcriptional activation domain (TAD), which recruits the MED25 subunit of the MEDIATOR complex, required for transcription initiation (Kidd et al., 2011;
Cevik et al., 2012). The N-terminus also possess a JAZ interaction Domain (JID), which differentiates MYC from other bHLH proteins, and is necessary for the interaction with the Jas domain of JAZ repressors (Chini et al., 2007; Fernández-Calvo et al., 2011; Kazan and Manners, 2013).
EVOLUTION
Land plants (or embryophytes) colonized earth around 470 million years ago (Szövényi et al., 2019). Phylogenetic evidence suggests that they evolved from an ancestral fresh-water charophycean algae from which they inherited biochemical, developmental and genetic attributes necessaries to make the transition to land (Bowman et al., 2017; Nishiyama et al., 2018; Szövényi et al., 2019). The development of plant life on land was one of the most significant episodes in earth history and current knowledge indicates that occurs most probably during early-middle Ordovician (Morris et al., 2018; Salamon et al., 2018; Szövényi et al., 2019). The evolution of land plants was associated to the origin of a multicellular sporophytic phase. The earliest forms were represented by small nonvascular bryophyte-like organisms (Renzaglia et al., 2014;
Salamon et al., 2018), they were unbranched, with terminal sporangia and simple rhizoid rooting systems and no leaves, that evolved later on to enable nutrient transport to support a large plant body (Bowman et al., 2017; Szövényi et al., 2019). Phylogenetic analysis shows that land plants consist on the clade of vascular plants (Tracheophytes) including lycophytes, monilophytes, gymnosperms and angiosperms, and a group of three lineages:
mosses, liverworts and hornworts, known as bryophytes (Szövényi et al., 2019) (Figure 4). The key evolutionary position of bryophytes makes them attractive models for the study of plant evolution. There availability of genome sequence, genetic studies, tools and resources for the moss Physcomitrella patens, and the liverwort Marchantia polymorpha make these plants suitable models.
However, in the last years, the low gene redundancy of M.polymorpha makes it even more interesting than P. patens (Bowman et al., 2017).
Figure 4: Phylogenetic relationships among green plants.
Chlorophytes and charophytes are green algae. Bryophytes and Tracheophytes comprise land plants (Embryophytes). Tracheophytes (vascular plants) include lycophytes, ferns, gymnosperms and angiosperms.
charophycean algae (i.e Klebsormidium flaccidum) suggesting that developmental mechanisms and their genetic regulators were present in the common ancestor of charophytes and land plants (Szövényi et al., 2019). However, plant evolution occurred together with an increased diversity of regulatory networks, including hormone signaling pathways (Bowman et al., 2019a, 2019b) each class of them with distinct evolutionary origin. For instance, F-box pathways (Auxin, JA, GA, strigolactones) originated in the ancestral land plant, whereas the two-component signaling pathways (ethylene, cytokinin) predate land plant evolution. Moreover, the ligands that activate these signaling pathways have co-evolved with their receptors within land plants (Bowman et al., 2019b).
MARCHANTIA POLYMORPHA
Marchantia polymorpha is the most common dioecious liverwort found around all continents except Antarctica. Is a member of the Marchantiopsida clade and is characterized by a thalloid gametophyte (Yamato et al., 2007; Berger et al., 2016; Bowman et al., 2017). Jean Marchant first named the genus Marchantia in 1713, and although its name has been widely used, many infraspecific taxa have been described, being three subspecies of M.polymorpha recognized: M. polymorpha subsp. polymorpha; M.
polymorpha subsp. montivagans; and M. Polymorpha subsp.
ruderalis (Shimamura, 2016). During nearly 200 years, Marchantia polymorpha has been used as a model system, using thallus, spores and gemmaes for investigating morphological and physiological responses to environmental factors (Bowman et al., 2016;
Shimamura, 2016). Marchantia occupy a pivotal position in land plant phylogeny being a link between other plant basal species with sequenced genome, such as the charophycean algae Klebsormidium
flaccidum, the moss Physcomitrella patens and the hornwort Anthoceros agrestis (Shimamura, 2016; Berger et al., 2016). This phylogenetic context together with its small genome size (280Mb) and simple life cycle, haploidy, ease of propagation and crossing, absence of ancient polypoidization and lack of gene duplication, converted Marchantia into a potential model organism (Shimamura, 2016; Schmid et al., 2018). In the last years, the development of transformation techniques (Ishizaki et al., 2008, 2015; Kubota et al., 2013) gene targeting (Ishizaki et al., 2013; Sugano et al., 2014a) and the JGI initiative to sequence the M.polymorpha genome (Bowman et al., 2017) corroborates Marchantia as a model system.
Moreover, as a dioecious organism that posses eight autosomes and one sex chromosome (X in female and Y in male), M.polymorpha has also been used for sex chromosome analysis (Yamato et al., 2007;
Bowman et al., 2016). Its life cycle alternates between two generations: the sporophyte and the gametophyte (Figure 5) (Shimamura, 2016; Schmid et al., 2018;) but it spends most of its live as an haploid life form (gametophyte) (Berger et al., 2016;
Shimamura, 2016; Delmans et al., 2017).
The male gametophyte forms antheridiophores, the reproductive structures that contains antheridia, which produce flagellated sperm (antherozoids). The female gametophyte forms archegoniophores that has archegonia, each holding a single egg cell. During sexual reproduction, sperms released from the antheridia swim towards the archegonia and fertilize the eggs. After fertilization, sporophyte is formed and hundred of thousands of haploid spores are pBowroduced through meiosis, germinating and developing into male (Y) or female (X) gametophytes concluding the life cycle. Asexual reproduction is possible in male and female gametophytes through the formation of gemmae in the gemma cups
on the dorsal side of the thallus, the gametophyte body that has a growing point at the apical notch. The transition from vegetative to reproductive growth is produced under long-day conditions supplemented with far-red light (FR) and develops gametangiophores on the thallus (Bowman et al., 2016; Ishizaki et al., 2016; Shimamura, 2016; Schmid et al., 2018).
Figure 5: Marchantia polymorpha life cycle (Schmid et al., 2018).
The study of M.polymorpha is also attractive because of the production of specialized metabolites, such as terpenoids and benzenoids, which posses pharmaceutical and agrochemical relevance. These compounds are accumulated in oil bodies in Marchantia, which used them for defense against pathogens and herbivores, and are also a clue to elucidate the strategy by which
liverworts adapt to terrestrial environment (Tanaka et al., 2016).
Moreover, the use of Marchantia as a new model plant has also proved the conservation of diverse hormonal signaling pathways between different lineages of land plants, encoding a complete set of land plant biosynthetic and signaling components, including TFs (Bowman et al., 2017, 2019b).
Analysis of Marchantia polymorpha genome and functional analyses of its COI1/JAZ co-receptor showed that the JA pathway first appeared in the common ancestor of land plants more than 450 million years ago (Bowman et al., 2017; Monte et al., 2018).
Moreover, sequences from all bryophyte lineages including hornworts, liverworts and mosses have revealed orthologs for all core components of this pathway such as CO1, JAZ, NINJA, TPL, and MYC (Bowman et al., 2017; Monte et al., 2018). Nevertheless, bryophytes neither synthesize nor respond to JA-Ile (Monte et al., 2018). Bryophytes thus have a conserved JA-Ile machinery lacking JA-Ile, and recently two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA) have been identified in M. polymorpha as bioactive ligands of MpCOI1 (Monte et al., 2018). The functional conservation of the co-receptor COI1/JAZ in bryophytes suggests that, evolutionarily, the JA pathway may have appeared during plant terrestrialization.
However, whether candidate MYCs in M. polymorpha are functionally conserved has not been addressed yet, nor has their origin from algal TFs.
In this work, we have characterized MYC-related TFs in the liverwort M. polymorpha. Phylogenetic analysis suggested that MYC function appeared in charophyte algae, and therefore precedes the appearance of any other component of the JA pathway. M.
polymorpha possesses two MYC genes, which are located in sex
chromosomes. Therefore, each plant bears only one MYC, MpMYCX in females and MpMYCY in males. Together, our results show that despite 450 million years of independent evolution, MYC TFs are functionally conserved between the bryophyte M. polymorpha and the eudicot A. thaliana. Genetic conservation of MYCs in one of the most basal plant lineages infers that MYC function was present in the common ancestor of land plants and evolved from a pre-existing MYC function in charophycean algae.
OBJECTIVES
OBJECTIVES
In the last few decades, Arabidopsis thaliana has been the predominant model system to study the JA signaling pathway.
Studies in Arabidopsis have uncovered the main components of this pathway and the hormone activating it. However, its conservation on other plant lineages has not been addressed so far. Recently, the bryophyte Marchantia polymorpha has emerged as a new model plant system for evolutionary studies due to its key phylogenetic position and low gene redundancy, among other interesting features. Recent results in our laboratory have shown the functional conservation of the COI/JAZ co-receptor complex and demonstrated that the ligand activating this co-receptor in Bryophytes is dinor-OPDA, instead of JA-Ile. These discoveries showed interesting differences in major components of the pathway that have been important to understand the evolution of jasmonates biosynthesis and signaling.
MYC TFs are direct targets of JAZs proteins in Arabidopsis thaliana and the main transcriptional regulators of JA-Ile responses. The main objective of this thesis was to study the functional conservation and evolution of the JA signaling pathway, studying the functional conservation of MYC transcription factors in Marchantia polymorpha.
The specific objectives are:
- Identification of homologue sequences to Arabidopsis thaliana MYC2, MYC3 and MYC4 in Marchantia polymorpha.
- Genetic and molecular analysis of MpMYCs function in Marchantia polymorpha.
MATERIALS AND METHODS
MATERIALS AND METHODS
Plant material and growth conditions
Marchantia polymorpha accession Takaragaike-1 (Tak-1; male) and Takaragaike-2 (Tak-2; female) were used as wild-type in this study.
Plants were grown on half strength Gamborg´s B5 (GB5) medium containing 1% agar under continuous light (50-60 µmol m-2 s-1) and 22°C. Lines generated in this study are listed in Supplemental Table 2.
Plants were subcultivated by cutting pieces of thalli including the notch (meristem) or by sowing gemmae from the gemma cups.
Gemmae were stored in vials containing 0.5 Gamborg’s B5 1% agar 1% sucrose at 4º C and darkness for several years. Gemmae used for confocal microscope observation were grown on liquid half strength Gamborg’s B5 liquid medium. Plants used for RNA extraction were grown on 0.5 Gamborg’s B5 medium containing 0.5% agar to facilitate removal from the agar plate when transferring the plants to 0.5 Gamborg’s B5 liquid medium for OPDA treatment.
For transformation assays (Ishizaki et al., 2008; Kubota et al., 2013), cut-thalli were grown in liquid 0M51C medium. Transformed
plants were plated on 0.5 Gamborg’s B5 1% agar medium plus hygromycin and/or chlorosulfuron. Cefotaxime sodium salt (also known as Claforan, Duchefa) was included in all the plates to prevent Agrobacterium tumefaciens growth. M. polymorpha plants used for crossing were grown on soil under continuous white light and covered with a lid to maintain humidity. Gametangiophores were induced under white light supplemented with far-red.
Ecotype Col-0 is the genetic background of Arabidopsis thaliana WT, mutants and transgenic lines used in this study. Seeds were sterilize by chlorine gas method (50 ml bleach and 3 ml HCl 37% for 3 h) and stratified at 4ºC in darkness during 2-3 days. Seedlings were grown on Johnson medium containing 0.55% agar and the indicated molecule under long day conditions at 22ºC.
Chemicals
OPDA (12-oxophytodienoic acid) used in this study was synthesized by Dr. Kosaku Takahashi (Agricultural University of Hokkaido) and (±)-Jasmonic acid was obtained from Sigma-Aldrich.
Plant transformation
M. polymorpha was transformed following the cut-thalli transformation method (Kubota et al., 2013). For A. thaliana transformation, the floral dipping method was used (Clough and Bent, 1998). T3 homozygous plants were selected in the appropriate antibiotics and these plants were used for further experiments.
Gene identification and phylogenetic analyses
M. polymorpha sequences were obtained from JGI and Marchantia community databases (http://phytozome.jgi.doe.gov and http://marchantia.info). Sequences were aligned using DiAlign
multiple analysis http://www.genomatix.de/cgi-bin/dialign/
dialign.pl). MpMYCs were PCR-amplified using gene specific primers and subsequently sequenced (Supplemental Table 3). Names of genes used in this study are listed in Supplemental Table 4.
Sequences of all green algae (Chlorophytes and Charophytes) from 1KP database (Matasci et al., 2014) were obtained and analyzed.
JID/TAD and bHLH domain sequences of Klebsormidiales (Klebsormidium nitens), Zygnematales (Cylindrocystis brebissonii, Roya obtusa, Spirogyra sp, and Mesotaenium braunii), Coleochaetales (Coleochaete scutata and Coleochaete irregularis) and Charales (Nitella mirabilis) were aligned using Dialign multiple analysis (Figure 6).
CRISPR/Cas9 mediated mutagenesis to obtain Mpmycy and Mpmycx mutants
One sgRNA for Mpmycy and one sgRNA for Mpmycx (Supplemental Table 3) were designed in the second exon after JID domain as in (Sugano et al., 2014b). Both sgRNA were cloned into pMpGE_En03 entry vector (Addgene plasmid #71535) carrying the Cas9.
Transformants were sequenced and cloned into pMpGE011 (Addgene #71537) destination vector. This vector was incorporated into Agrobacterium tumefaciens GV3101, which was used to transform Tak-1 and Tak-2 plants, respectively. Transformants were selected on chlorosulfuron, genotyped and sequenced to identify the mutations.
Fertility assays
Plants used for crossing were grown on soil under continuous white light and supplemented with far-red to induce gametangiophores.
30 days after crossing, spores of each cross were harvested and
stored at -80ºC. Quantification of spore number was performed using a Neubauer Chamber and Leica Olympus DP70 using 10x and 20x objective.
Microarray analysis
Total RNA from either 15-day-old Mpmycs and WT gemmalings treated with OPDA for 2 h in liquid 0.5 GB5 medium or wounded (2 h) 21-day-old gemmalings grown in 0.5 GB5 1% agar was extracted using RNeasy plant Mini kit (Qiagen). DNase on-column digestion (Qiagen) was performed to remove genomic DNA contamination and quality was assessed in a Bioanalyzer 2100 (Agilent), according to the manufacturer’s instructions. Two hundred nanograms were amplified and Cyanine 3- (Cy3-) and Cy5-labeled using Two-Color Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer's recommendations. Labeled cRNAs were purified with RNeasy columns (Qiagen) and RNA yield and Cy3/Cy5 incorporation measured in a Nanodrop spectrophotometer (Nanodrop Technologies).
Probe preparation, hybridization and scanning were performed as described for Agilent's 8x60k microarrays (Two-Color Microarray- Based Gene Expression Analysis, Agilent Technologies) in Monte et al., 2018. Images for Cy3 and Cy5 channels were captured in an Agilent DNA Microarray Scanner at a resolution of 2 μm, and spots quantified using Agilent Feature Extraction Software.
Correction and normalization of expression data were performed using the methods "normexp" and loess in LIMMA, respectively (Smyth, 2004). Differentially expressed genes were evaluated by the non-parametric algorithm 'Rank Products' available as RankProd package at Bioconductor (Hong et al., 2006). We considered as differentially expressed those genes with LogRatio greater than 1 or
lower than -1 and the expected false discovery rate (FDR) less than 5%. Every gene was represented by at least three oligonucleotide probes, we obtained a table of gene expression values across all the experiments, and simplified it by selecting just one probe per gene showing the lowest FDR value. Clustering of genes was performed using K-Means with euclidean distance (Soukas et al., 2000) in Multi Experiment Viewer (http://mev.tm4.org/).
Herbivory assays
Marchantia polymorpha gemmae were grown on half Gamborg’s medium (Duchefa) containing 1% agar in continuous light (20°C, 120 μmol m-2 s-1) for seven days before being transferred to soil (three per pot). Thalli were then grown for five weeks in a growth chamber (21°C, 10/14 h light/dark cycle, 100 μmol m-2 s-1) using a lid to cover the tray and maintain high humidity.
Insect feeding assays on WT, Mpmycy mutants and 35S:MpMYCX- Citrine/Mpmycy were performed after 6-week. For each biological replicate, a total of 40 freshly hatched S. littoralis larvae (eggs obtanin form syngenta) were placed on plants of each genotype in transparent plexiglas boxes. After 10 days, larval weight was assessed with a precision balance Mettler-Toledo (Greifensee, Switzerland). The experiment was repeated four times independently with similar results.
Terpene extraction and quantification
Four biological replicates per genotype and treatment were collected after 3 days of herbivory and flash frozen in liquid nitrogen. Each sample was ground in liquid nitrogen with mortar and pestle. 1 ml of dichloromethane was added to 50 mg of ground tissue and the solution was stired for 4h at RT. After cooling down
the samples to 5°C for 1h, samples were centrifuged and 90 µl of supernatant was transferred into GC/MS glass vials for analysis.
To each sample was added 10 µl of a 10 µg/mL solution of internal standard ((-)-isopulegol, CAS 89-79-2, Sigma-Aldrich, St. Louis, MO). The GC-MS system used was an Agilent 7890B coupled with a 5977A MSD mass spectrometer, both controlled by Mass Hunter B.07.02 software (Agilent Technologies, Santa Clara, CA). 2 µL of solution was injected in splitless mode on a 30 m x 0.25 mm Rxi- 5Sil MS capillary column with 0.25 µm film thickness (Restek, Bellefonte, PA). The carrier gas was helium delivered at a flow rate of 1 mL/min. The following temperature program was used: starting temperature 60°C, which was kept for 3 min, then raised to 120°C at a rate of 10°C /min and then 5°C/min up to 280°C. Total runtime was 41 min. The temperatures for inlet, transfer line, MS source and MS quadrupole were 270, 280, 230 and 150°C, respectively.
The mass spectrometer was set in electron ionization mode using a scan time of 4 scans/sec and covering a mass-to-charge (m/z) range from 40 to 400. A solvent delay of 7 min at the beginning of the run was applied. Compounds were identified based on mass, fragmentation patterns and comparison with the NIST (National Institute of Standards and Technology, Gaithersburg, MD) database and authentic standards: thujopsene (CAS 470-40-6, Sigma- Aldrich), β-Chamigrene (CAS 18431-82-8, Sigma-Aldrich), and cuparene (CAS 16982-00-6, Sigma-Aldrich).
Cloning
MpMYCY (MapolyY_B0018.1) and MpMYCX (Mapoly0018s0019.1) were amplified respectively from Tak-1 and Tak-2 cDNA with High Fidelity polymerase (Roche), using Gateway specific primers (Supplemental Table 3). Subsequently, both were cloned into
pDONR207 through BP reaction, and LR reaction (Invitrogen) were performed to clone them into different vectors (Supplemental Table 2 and 5). MpJAZ and AtJAZs cloned into pGILDA, pGBKT7 and pGADT7, respectively, were already available in the laboratory (Chini et al., 2009; Monte et al., 2018, 2019; Supplemental Table 5). MpMYCY, MpMYCX and AtMYC2 were cloned into pMpGWB106 for constitutive expression with 35S promoter and Citrine C- terminal fusions (Supplemental Table 2). Transgenic overexpression plants were selected based on their resistance to hygromycin.
MID (MYC interaction domain) region of MpMED25 (Mapoly0022s0114) was amplified using specific primers (Supplemental Table 3) and cloned into yeast two-hybrid vector.
AtMED25 was provided by Chen et al., (2012) (Supplemental Table 5).
Protein expression, purification and determination of DNA binding motifs
Maltose-binding-protein (MBP)-fused proteins MpMYCY and MpMYCX were expressed in E. coli BL21 and purified as previously described (Fonseca and Solano, 2013).
To determine DNA-binding specificities, MBP-MpMYCY and MBP- MpMYCX fusion proteins were hybridized to protein binding microarrays (PBM) as previously described (Godoy et al., 2011).
Pull-down assays
Protein extraction and subsequent pull-down assays between MpJAZ and MpMYCs were performed as previously described (Fonseca and Solano, 2013; Fonseca et al., 2009b). A. thaliana extracts over-expressing MpJAZ-GFP were incubated with
recombinant MpMYCs MBP-fused proteins (expressed in E. coli BL21) for 1 h at 4ºC in a daisy-wheel rotator. Samples were washed with 500 μl pull- down buffer for 3 min twice. Then samples were resuspended in 35 μl pull-down buffer, and one fraction was used for immunodetection with anti-GFP-HRP antibody (MACS). Another fraction was used for Coomassie Blue Staining as loading control.
Confocal microscopy
Gemmae of transgenic plants expressing either 35S:MpMYCY- Citrine or 35S:MpMYCX-Citrine, and WT (Tak-1 and Tak-2) plants, were cultured on 0.5 GB5 liquid medium for two days. Citrine signal was visualized in a Leica TCS SP8 laser scanning confocal microscope (excitation at 510 nm; 25% power and emission band at 580 nm). A 20X objective was used to acquire Z-series section images (1024 x 1024 resolution). Images were acquired and processed using Leica Application Suite (LAS-AF, v.2.7.3) and imageJ was used for obtention of merged pictures by Z projection.
Western blot analysis
M.polymorpha or A.thaliana plants were harvested in frozen liquid nitrogen and homogenized in Laemmli SDS-PAGE protein loading buffer. The extracts were boiled at 95°C for 5 min and kept in ice. A 20ul volumen sample was run into SDS-PAGE gel and transferred to nitrocellulose membrane (Bio-Rad).
Citrine- or GFP-tagged proteins expressed in the different transgenic M. polymorpha or A. thaliana lines (Supplemental Table 2) were immunodetected with anti-GFP-HRP antibody (MACS) and actin was immunodetected with anti-actin (Sigma-Aldrich) and anti- mouse-HRP (GE Healthcare; Chico et al., 2014).
Light Treatments
Gemmae of transgenic plants expresssing 35S:MpMYCY-Citrine or 35S:MpMYCX-Citrine, and WT (Tak-1 and Tak-2) plants were grown in white light/dark cycles (WL/D, 16/8) for 10 days and exposed for 24 h to dark. Samples were harvested in liquid nitrogen and immunodetection by western blot were performed as described in (Chico et al., 2014).
Yeast two-hybrid assays
Yeast two-hybrid assays using the LexA system were performed as in (Monte et al., 2018) to test interaction between MpJAZs and MpMYCs (Figure 10A). Corresponding vectors pB42AD (bearing the activation domain, AD) and pGILDA (for fusions to the binding domain, BD) were used. Yeast strain used was EGY48 (p8opLacZ).
For MpMYCs-AtJAZ and MYC-MED interactions (Figure 10C and 24), yeast two-hybrid assays as in (Chini, 2014) were performed using pGADT7 (bearing the activation domain, AD) and pGBKT7 (bearing the binding domain, BD) vectors. Yeast strain used was AH109.
qPCR analysis
Plant total RNA purification mini kit (Favorgen®) was used for RNA extraction using five gemmalings per sample. cDNA was synthesized from one microgram of RNA using High-Capacity cDNA Reverse Transcriptase (Applied Biosystems). PCR were performed on a 7500 thermocycler using SYBR-green or on a 7900HT sequence detection system (Applied Biosystems) using 5x HOT FIREpol EvaGreen® qPCR Mix Plus (ROX; Solis biodyne). Expression of MpMYCX, MpMYCY, MpDIRIGENT-LIKE PROTEIN, MpPATATIN-LIKE PROTEIN 5, MpbHLH62 was analysed using MpACT as housekeeping gene.
Statistical analysis
Statistical significance based on Student’s t-test analysis was calculated using Excel (Microsoft) and ANOVA using agricolae package from Rstudio software (https://www.rstudio.com).
Experimental contributions
I, María Peñuelas Hortelano performed all the experiments except the microarray design, hybridization and data analysis (Gloria García-Casado and José Manuel Franco, Genomics Unit, CNB-CSIC, Madrid), hervibory assays and sesquiterpenes quantification (Fabian Schweizer, Philippe Reymond and Armelle Vallat, from Department of Plant Molecular Biology, University of Lausanne and Neuchatel Platform of Analytical Chemistry, Institute of Chemistry, Faculty of Sciences, University of Neuchâtel, Switzerland, respectively). Gemma Fernandez Barbero helped me with Pull Down assays, and Isabel Monte (Plant Molecular Genetics, CNB-CSIC, Madrid) teach me the use of Marchantia polymorpha tecniques and advise me with experimental procedures.
RESULTS
RESULTS
MYC proteins predate plant terrestrialization
MYC proteins belong to the bHLH family and differentiate from other bHLHs by the presence of a JAZ-Interaction-Domain (JID;
(Fernández-Calvo et al., 2011)). We have previously identified two sequences encoding MYC-related bHLH TFs in the genome of M.
polymorpha that we named MpMYCX and MpMYCY because of their location in sex chromosomes (Bowman et al., 2017). In an attempt to reconstruct the phylogenetic history of the MYC subfamily we used AtMYC2, MpMYCX and MpMYCY to identify JID-containing bHLH proteins in green algae using BLAST and the 1KP database (Matasci et al., 2014). Alignment of the identified sequences by DiAlign (genomatix.de/cgi-bin/dialign/dialign.pl) revealed the presence of the JID, TAD and bHLH domains in proteins from different clades of charophycean algae, whereas no significant similarity was identified in chlorophytes (Figure 6).
B
Figure 6: Conservation of the JID, TAD and bHLH in the green lineage. (A) Multiple sequence alignment of JID and TAD domains from MYCs transcription factors in green algae species (Klebsormidium nitens, Cylindrocystis brebissonii, Roya obtusa, Spirogyra sp, Mesotaenium braunii, Coleochaete escutata, Coleochaete irregularis, Nitella mirabillis), M. polymorpha and A. thaliana. (B) Multiple sequence alignment of bHLH domain from MYCs transcription factors in same species than in (A).
In the case of Klebsormidium nitens the only protein identified contained a JID and bHLH domains but lacked the TAD. Although MYC proteins have not been identified in Chara braunii (Nishiyama et al., 2018), we found MYC relatives in other Charales such as Nitella mirabilis (Figure 6 and 7). These results suggest that MYC TFs precede the appearance of the JA signalling pathway that occurred during land colonization by plants more than 450 million years ago (Bowman et al., 2017). The fact that MYCs are present in most but not all charophycean lineages suggests that they likely
K.nitens C.brebissonii R.obtusa Spirogyra sp.
M.braunii C.scutata C.irregularis N.mirabilis MpMYCY MpMYCX AtMYC2
1 JID 61
K.nitens C.brebissonii R.obtusa Spirogyra sp.
M.braunii C.scutata C.irregularis N.mirabilis MpMYCY MpMYCX AtMYC2
TAD
121
K.nitens C.brebissonii R.obtusa Spirogyra sp.
M.braunii C.scutata C.irregularis N.mirabilis MpMYCY MpMYCX AtMYC2
1 51
bHLH
evolved during charophycean diversification (between 850 and 500 million years ago (Knauth and Kennedy., 2009) and might have been co-opted for the JA signalling pathway during land plant colonization.
Figure 7: Simplified cladogram showing the phylogenetic relationships between the major groups of plants. Grey shade and green lines indicates the presence of JID and TAD domain in MYC sequences. Discontinuous line indicates partial domain conservation (presence of JID and bHLH but absence of TAD).
However, sequence conservation does not guarantee functional conservation and to challenge this co-option hypothesis we functionally characterized MpMYCs to find out if MpMYCX and MpMYCY are required for dn-OPDA responses in M. polymorpha and, therefore, have a similar function to Arabidopsis MYCs orthologs.
Nuclear localization of MpMYCs and interaction with MpJAZ MYC2 is a nuclear-localized protein in Arabidopsis (Lorenzo et al., 2004). In order to test the subcellular localization of MYC proteins in M. polymorpha, sequences of both MpMYC genes were amplified by PCR and cloned. Consistent with their predicted localization in
Chlorophytes Mesostigmatales Chlorokybales Klebsormidiales ZygnematalesColeochaetales
Charales
Charophytes
Hornwort Liverwort Moss Bryophytes
Tracheophytes
sex chromosomes, MpMYCY could only be amplified from wild-type M. polymorpha Tak-1 accession (male) cDNA and MpMYCX only from Tak-2 accession (female) cDNA (Figure 8).
Figure 8: MpMYCs are located in sexual chromosomes.
Electrophoretic image of a specific fragment of MpMYCY or MpMYCX PCR amplicons using genomic DNA from Tak-1 or Tak-2. MpMYCY was amplified only in Tak-1 (male) and MpMYCX only in Tak-2 (female).
Subcellular localization was analyzed in transgenic plants expressing MpMYCY-Citrine or MpMYCX-Citrine in their corresponding WT background (35S:MpMYCY-Citrine in Tak-1 and 35S:MpMYCX-Citrine in Tak-2). Confocal microscopy of the resulting lines confirmed a conserved nuclear localization of both MpMYCs (Figure 9).
Figure 9: MpMYCs are nuclear proteins. MpMYCs nuclear localization by confocal imaging of 2-day-old 35S:MpMYCY-Citrine and 35S:MpMYCX-Citrine gemmalings and WT (Tak-1 and Tak-2). Close-ups (5x) of the nuclei are shown on the right. Scale bars, 100 mm.
In Arabidopsis MYC proteins interact with JAZ through their JID domain (Figure 3). To test if MpMYCY and MpMYCX are MpJAZ targets we tested their physical interaction using yeast two-hybrid assays. As shown in Figure 10A, both MpMYC proteins were able to interact with full-length MpJAZ, but not with a truncated version lacking the Jas domain (MpJAZΔJas), which is required for MYC interaction in Arabidopsis JAZ proteins (Chini et al., 2007).
MpMYC-MpJAZ interaction was confirmed by pull down assays (Figure 10B). We also tested if MpMYCs could interact with AtJAZs.
Out of the 13 Arabidopsis JAZ proteins, we tested 11, and in all
cases (with the possible exception of AtJAZ7 and MpMYCX) the interaction with both MpMYC proteins was detected (Figure 10C).
These results indicate that the role of the JID and Jas interaction domains is fully conserved between MYC and JAZ proteins from M.
polymorpha and A. thaliana, and suggests that MpMYCs are MpJAZ targets in M. polymorpha.
Figure 10: MpMYCs interact with JAZs. (A) Yeast two-hybrid assays between MpMYCs (fused to AD, pB42AD) and MpJAZ (fused to BD, pGILDA). Yeast was grown on SD medium with glucose (Glu) (–Ura-Trp- His) to verify the presence of both plasmids or with galactose / raffinose (Gal/Raf) (-Ura-Trp-His + X-Gal) to test the protein interactions. Blue color indicates positive proteins interaction. (B) Immunoblot (anti-GFP antibody) of recovered MpJAZ-GFP (from 35S:MpJAZ-GFP Arabidopsis extracts) after pull-down reactions using recombinant MpMYCs-MBP protein. Bottom, Coomassie blue staining of the input quantity of recombinant proteins used. (C) Yeast two-hybrid assays between MpMYCs (fused to AD, pGADT7) and AtJAZs (fused to BD, pGBKT7). Yeast was grown on selective media lacking Leu and Trp (-2) to verify the presence of both plasmids or in selective media lacking Ade, His, Leu and Trp (-4)
Constitutive expression of MpMYCs confers hypersensitivity to OPDA
Constitutive expression of AtMYC2 in Arabidopsis promotes JA hypersensitivity (Lorenzo et al., 2004). To test the effect of MpMYC overexpression in M. polymorpha, we analyzed the response to OPDA of the 35S:MpMYCY-Citrine and 35S:MpMYCX-Citrine transgenic lines and compared it with that of WT Tak-1 or Tak-2 plants and the OPDA-insensitive Mpcoi1-1 mutant. As shown in Figure 11 and Supplemental Figure 1, the transgenic lines grown in mock plates were slightly smaller than the WT, and growth- inhibition caused by OPDA (5 µM) was more pronounced than in the WT controls.
Figure 11: Overexpression of MpMYCs confers hypersensitivity to OPDA. (A) and (B) Growth inhibitory effect of OPDA (5 µM) on 12-day- old M. polymorpha gemmalings of WT plants (Tak-1, Tak-2) and 35S:MpMYCY-Citrine, 35S:MpMYCX-Citrine transgenic lines. Mpcoi1-1 was included as a control for OPDA insensitivity. Experiment repeated 3 times with similar results. n=12-15 plants. Scale bar, 1 cm. (C) Plant area of WT (Tak-1, Tak-2), Mpcoi1-1 and 35S:MpMYCY-Citrine, 35S:MpMYCX- Citrine lines grown in control and OPDA concentrations as in (A). Data shown as mean ± S.D. Statistical analysis was done by ANOVA; letters
