Plant PARPs, PARGs and PARP-like proteins

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Plant PARPs, PARGs and PARP-like proteins

Running title: Poly(ADP-ribos)ylation in plants

Julia P. Vainonen*, Alexey Shapiguzov*1, Aleksia Vaattovaara, Jaakko Kangasjärvi2

Plant Biology Division, Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland

Corresponding author:

Jaakko Kangasjärvi

Plant Biology Division

Department of Biosciences

University of Helsinki

POB 65 (Viikinkaari 1)

FI-00014 Helsinki Finland

Tel: +358 2941 59444

Fax: +358 2941 59552

Email: [email protected]

* These authors contributed equally to the work

1Permanent address: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya street, 35, 127276 Moscow, Russia 2Second affiliation: Distinguished Scientist Fellowship Program, College of Science, King Saud University, Riyadh, Saudi Arabia

mailto:[email protected]

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Abstract: Poly(ADP-ribos)ylation, originally described as a mechanism of DNA break repair, is now considered as part of a complex regulatory system involved in dynamic reorganization of chromatin structure, transcriptional control of gene expression and regulation of metabolism. In plants poly(ADP-ribos)ylation has received surprisingly little attention. It has been implicated in abiotic and biotic stress responses, cell cycle control and development; however the molecular mechanisms and proteins involved are largely unknown.

In this review we summarize current knowledge on plant PARP, PARG and PARP-like domain containing proteins and discuss their possible roles in plant development, innate immune responses, programmed cell death and stress responses in general. The genome of the model plant Arabidopsis contains three genes encoding PARP proteins, two of which have been shown to be active PARPs, and two genes encoding PARG proteins, one of which was shown to possess enzymatic activity. In addition, SROs (Similar to RCD One) represent a plant specific family of proteins containing a PARP-like domain. Although bioinformatics and biochemical data suggest that the PARP-like domain in SRO proteins does not have PARP activity, these proteins play a significant role in stress response as revealed by mutant analysis. SRO proteins interact with transcription factors involved in various stress and developmental responses and are suggested to serve as hubs in many signaling pathways. Altogether current data imply that poly(ADP-ribos)ylation plays significant regulatory role in many aspects of plant biology.

Keywords: poly(ADP-ribos)ylation, PARP, PARG, SRO, RCD1, Arabidopsis thaliana, transcriptional regulation

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1. Introduction

Plants are constantly exposed to a variety of abiotic and biotic stresses that negatively affect their growth and productivity. The most immediate chemical responses of plants to environmental cues generally involve changes in the location and activity of pre-existing proteins via posttranslational modifications (PTMs) such as phosphorylation, acetylation, or ubiquitination. The percentage of genes in the genome of the model plant Arabidopsis thaliana encoding protein kinases/phosphatases and components of the ubiquitin-26S proteasome system (UPS) is several times higher than in non-plant eukaryotes [1] which highlights the importance of protein posttranslational processing for plants. While the role of reversible protein phosphorylation and protein degradation by the UPS in plant development and stress responses is well documented, the description and information on other PTMs in plants is very limited.

Poly(ADP-ribos)ylation (PARylation) is a reversible PTM which regulates protein function in a variety of cellular processes, including chromatin remodeling, transcription, and programmed cell death in cross-talk with phosphorylation cascades and calcium signaling [2-4]. Originally PARylation has been described decades ago as a DNA damage-dependent protein modification required for efficient repair of DNA breaks and genome stability. However, intensive functional studies of PARylation in animal systems have expanded the regulatory potential of PAR far beyond the initial assumptions [5-7].

Attachment of poly(ADP-ribose) (PAR) to acceptor proteins is catalyzed by PAR polymerases (PARPs). The enzymes use NAD+ as a substrate to transfer ADP-ribose moieties to Glu, Asp or Lys amino acid residues of acceptor proteins with subsequent release of nicotinamide. The resulting ADP-ribose polymer is linear or branched polyanion of variable size (2-200 ADP-ribose units) [8]. Accordingly, the enzymes able to attach only a single unit of ADP-ribose are called mono (ADP-ribosyl) transferases (mARTs).

PARylation is a reversible, transient modification. PAR glycohydrolase (PARG) degrades covalently attached polymer releasing free PAR. PAR is a known cell death signal in mammalian cells [9]; however, there is no experimental evidence available whether this is the case for plants. PAR turnover in vivo is very fast with a half-life of a few minutes [8] which, together with the flexible chemical nature of PAR, suggests potential regulatory role for this polyanion. PAR is now recognized as an important signaling molecule which regulates protein function via two major mechanisms: covalent attachment to an acceptor protein and strong non-covalent interactions where the size and shape of PAR might determine selectivity of the interacting proteins [6, 7, 10].

In contrast to mammalian systems, surprisingly little is known about PARylation in plants. Despite the first documentation of PARP activity in plant nuclei already in the late 1970’s [11, 12], many studies on mechanisms of PARylation and its relevance for plant physiology have been conducted only recently. Recent data suggest that PARylation plays an

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important regulatory role in several physiological processes in plants. In this review we describe structure and function of plant PARP- and PARG-domain containing proteins and discuss their role in stress and developmental responses.

2. Phylogenetic analysis of PARP and PARG proteins in plants

We analyzed PARP domain protein sequences from 29 species of plants with sequenced genomes (Table S1). The sequences were analyzed with PASTA [13] and protein models containing PARP domains were recognized with HMMER and BLAST searches against the selected genomes. From the eukaryotic microalga Coccomyxa subellipsoidea and a unicellular green alga Ostreococcus lucimarinus no protein models containing PARP domains were found. Protein models from the green algae Chlamydomonas reinhardtii and Volvox carteri and the related protein model from a bryophyte Physcomitrella patens (classified as Clade 6 genes by Citarelli et al. [14]) produced the outgroup for the main clades of PARPs and SROs. Phylogenetic analyses revealed that PARPs form three phylogenetic groups in plants (PARP1, PARP2, PARP3; Fig. 1). These three groups belong to Clade 1 in the classification presented by Citarelli et al. [14]. The occurrence of PARPs is conserved throughout the plant kingdom; all the land plant species shown in Fig. 1 and Table S1 have at least one gene in each of the three groups. Proteins of all three groups have specific domain structures, but in all groups the proteins contain the WGR domain (that is proposed to bind to nucleic acid), the PARP regulatory and the PARP catalytic domains.

The plant PARP1 proteins in the 29 species analyzed here (Supplementary Fig. S1), including the lower vascular plants Selaginella and Physcomitrella, have a conserved domain structure that is similar to human PARP1. In phylogenetic tree human PARP1 also clusters close to plant PARP1 clade (Fig. 1). PARP1 is represented by two zinc-finger domains (involved in DNA binding), PADR1 domain of unknown function, BRCT domain (implicated in the binding of phospho-peptides, proteins, DNA and PAR), WGR domain, PARP regulatory and PARP catalytic domains. Most of the plant species analyzed here have only one PARP1 gene except for Glycine max and Theobroma cacao which contain two PARP1 genes, most likely as a result of the recent whole genome duplication in G. max and a tandem duplication of the gene in T. cacao.

The PARP2 proteins (Supplementary Fig. S2) contain SAP domains (SAF-A/B, Acinus and PIAS, putative DNA or RNA binding domains found in diverse nuclear and cytoplasmic proteins) in addition to the common domains. The number of SAP domains seems to be poorly conserved. In Selaginella there are two PARP2 genes, one of which encodes a protein that lacks a SAP domain, while the product of the other gene has two SAP domains. In Physcomitrella genome two gene models with two or four SAP domains are predicted for PARP2 (Phpat.022G047900) (Supplementary Fig. S2). The duckweed Spirodela polyrhiza PARP2 gene model (Spipo0G0091200) has only one SAP but this gene model is partial due to missing sequence in the genome in the location of the gene. In the legume family (including Glycine max and Medicago truncatula) the progressive loss of PARP2 SAP domains is observed with predicted proteins having only one SAP domain or no SAP domains at all (Supplementary Fig.

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S2). The monocot species rice and Sorghum have two copies of PARP2 gene. This seems to be a result of recent tandem duplication as the genes are located next to each other in the genomic sequences. Similarly to legumes, one of the monocot PARP2 genes encodes a protein with no SAP domains. Among the Solanaceae species, eggplant has only one PARP2 gene with two predicted SAP domains while both tomato and potato have two predicted PARP2s; one with three and the other with no SAP domains. In both species PARP2 genes are located next to each other, thus the duplication has most likely happened before the two species were separated (Supplementary Fig. S2). In Aquilegia coerulea genome three predicted PARP2 genes can be found in repeat configuration; all of them contain three SAP domains, but one of them is missing the WGR and the PARP regulatory domains. Populus has two predicted PARP2 genes, one with two and the other with one SAP domain. Again, these genes are located next to each other in the genome sequence suggesting that the pairs have arisen via tandem duplication.

PARP3 proteins resemble PARP1 without the zinc-finger domains. For this reason the two groups have been placed to the same clade in earlier phylogenetic analyses [14]. The split of the two groups is already observed in mosses (Physcomitrella) (Supplementary Fig. S3). It is not clear whether PARP3 proteins have lost zinc-finger domains or PARP1 proteins have gained them after the divergence to these two groups. Most of the sequenced plant species have only one PARP3 with the exception of the Solanaceae lineage where a duplication event has resulted in two PARP3 genes (in eggplant, potato, and tomato) (Supplementary Fig. S3).

In addition to the three described groups of PARPs, higher plants have a plant-specific family of proteins containing PARP-like domains, called the SRO proteins (Fig. 1). The name SRO comes from “Similar to RCD One” and refers to RCD1 (“Radical-induced Cell Death 1”) protein of Arabidopsis [15] (see below). SROs form two major phylogenetic groups with Group I encoding proteins that contain an N-terminal WWE domain when the Group II proteins are shorter and lack the WWE domain [14, 15]. In this phylogenetic analysis with more data compared to previous studies the two main groups do not produce two separate clades (Fig. 1) as in previous studies [14, 15] but the subgroups (Ia-c and IIa-b) have the same genes as in previous analyses.

Group I contains 3 subgroups. Group Ia contains of both basal and higher angiosperms (Supplementary Fig. S4). Most of the eudicot species have at least two genes in this sub-group. In monocots the phylogeny indicates duplication event in monocot lineage after duckweed and, in addition, independent duplication in maize (which has three genes in Group Ia). The Group Ib contains only monocots (Supplementary Fig. S5). In addition to angiosperms, Group Ic contains also one gene from Selaginella and three from Physcomitrella (Supplementary Fig. S5). Brassicaceae and Fabaceae species do not have genes for Group Ic SRO proteins. Largest expansion in the SRO genes has happened in the Solanaceae species, leading to, for example, five predicted Group Ic genes in tomato. There have also been duplication events in maize and in cacao in Group Ic.

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SRO2 genes are divided to two subgroups, IIa and IIb (Fig. 1 and Supplementary Fig. S6), after Amborella and duckweed (Spirodela polyrhiza). The duckweed is the only monocot that contains SRO2-like gene, so it is possible that the SRO2s have been lost in the monocot lineage after duckweed. In both groups there are only eudicots and there have been a duplication in lineage to Brassicaceae in both groups (in Group IIa SRO2 and SRO3 and in IIb SRO4 and SRO5, also Capsella and Lyrata have these duplications). There has been expansion in Populus trichocarpa in group IIa genes which has 4 genes.

Among plant genomes analyzed here, PARG genes were only found in land plants where they form a single clade (Fig. 2). Human PARG was used as an outgroup for the plant PARGs. At the bottom of the clade there are two gene models from the primitive land plants Physcomitrella and Selaginella. In monocots only Brachypodium distachyon has several PARG genes but in the gene models for PARGb, PARGc and PARGd the PARG domain seems to be truncated. There has been duplication in lineage to Solanaceae. In the lineage to Brassicacea there seems to have happened two duplication events, but from the later duplication one of the genes has turned to a pseudogene in Arabidopsis thaliana (At2g31860 which was excluded from the analysis). Similarly, PARG1b in Glycine max might have become a pseudogene. Presence of the PARG genes is conserved in land plants as those genes can be found from all the study species starting from Physcomitrella.

3. PARPs and PARGs in plants 3.1. PARP activity in plants

Eukaryotic organisms, with the exception of yeast, express several different proteins that contain the conserved PARP catalytic domain. For example, in human seventeen PARPs or PARP-like proteins have been described [6] (Fig. 3). In this review the PARP nomenclature is used (for the correspondence to the recent ARTD nomenclature in animals, see [16]). PARP proteins are linked to several processes such as DNA damage repair, transcriptional regulation, chromatin remodeling, circadian clock, metabolism, and the proteasome functions [4-6]. Some of the proteins are mono ADP-ribosyl transferases (mARTs) and for some, neither PARP nor mART activity has been shown [6, 16]. Plants have relatively few PARP protein families compared to animals (Fig. 3). The Arabidopsis genome contains three genes encoding PARPs: PARP1 (At2g31320), PARP2 (At4g02390), and PARP3 (At5g22470) according to the TAIR database nomenclature. AtPARP1 (originally named PARP2) shows high structural similarity with human PARP1 (HsPARP1), whereas AtPARP2 (originally named PARP1) and AtPARP3 resemble human PARP3 (HsPARP3). Since under standard growth conditions AtPARP3 is expressed mostly in developing seeds [17], studies of PARylation in Arabidopsis have been focused mainly on AtPARP1 and AtPARP2.

In plants, incorporation of radioactive NAD+ to proteins and PARylation of histones were first detected in wheat nuclei and nuclei of cultured tobacco cells already 3.5 decades ago

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[11, 12], whereas enzymes responsible for PARylation were first characterized 15 years ago in Arabidopsis, maize, and soybean [18-20]. Both AtPARP1 and AtPARP2 localize to the nucleus and in the presence of activated DNA transfer ADP-ribose moieties from NAD+ to themselves (automodification) and to acceptor proteins in vitro and in vivo [21]. Similarly to animal PARPs, plant PARP activities are inhibited by established PARP inhibitors such as 3-aminobenzamide (3AB) and 3-methoxybenzamide (3MB) which have been used in many studies [18-21]. Interestingly, opposite to animal PARPs, AtPARP2 from Arabidopsis had higher enzymatic activity than AtPARP1 [21]. The Arabidopsis parp and parg null mutants are viable and have normal growth without any adverse or abnormal secondary effects that would hamper their use to analyze the physiological role of PARylation in plants. This, in addition to chemical PARP inhibitors, provides tools to understand protein PARylation and regulatory mechanisms in plants in diverse biological processes at the whole organism level.

3.2. PARPs and abiotic stress response

The same way as in animals, plant PARPs play a role in plant DNA repair processes. In Arabidopsis AtPARP1 and AtPARP2 genes were rapidly induced by genotoxic agents [22], ionizing radiation (IR) and reactive oxygen species (ROS) [23]. AtPARP1 transcripts accumulated in all plant organs after exposure to IR; however, the protein levels increased only in tissues with actively dividing cells [23]. This cell type-specific accumulation of AtPARP1 protein in response to DNA damage is compatible with a role for the AtPARP1 protein in the maintenance of DNA integrity during replication. Accordingly, parp1 and parp2 mutants were slightly sensitive to DNA damage, and this sensitivity was increased in the double mutant where both PARP proteins were absent [24, 25].

Similarly to animal PARPs, plant PARPs are also involved in the programmed cell death (PCD). Depending on stress severity, the outcome of PARP activity might be diametrically opposite. In soybean cell cultures under mild stress caused by low concentrations of H2O2, PARP1 overexpression promoted DNA repair and inhibited cell death, whereas under severe stress caused by high H2O2 doses PARP overexpression increased cell death [26]. This observation suggests a model where overactivation of PARP under severe stress leads to increased NAD consumption and depleted NAD+ pool, resulting in ATP starvation and eventually in cell death. Addition of PARP inhibitors or expression of antisense PARP1 reduced the degree of cell death triggered by high doses of H2O2 [26]. PARP inhibitors also protected tobacco cell suspensions from heat-shock induced PCD [27]. However, the model where PARP activity leads to cell death through ATP depletion and energy collapse has recently been challenged in mouse cell culture system by the observation that PARP1 affected glycolysis and mitochondrial functions without observable NAD+ depletion [28] . This suggests novel roles for the enzyme, but the significance of this finding for understanding PCD in plants is unknown.

Abiotic stresses such as drought, cold, high light or heat led to activation of plant PARPs [29]. Despite the fact that under standard growth condition the Arabidopsis AtPARP3 is expressed in developing seeds [17], AtPARP3 was the most responsive PARP gene induced in

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leaves by several stress treatments such as high light, methyl viologen or high salt treatment [30]. As described above, this activation does not necessarily lead to increased plant viability. On the contrary, silencing of AtPARP1 and AtPARP2 in Arabidopsis or PARP silencing in oilseed rape plants improved plant tolerance to drought, high light or heat stress, and this resistance was correlated with reduced level of PAR [31]. The mechanism of the resistance is poorly understood. One possibility is the lower NAD+ consumption and improved energy use efficiency that would allow the NAD+ and ATP pools to be maintained and avoid the activation of the cell death pathways. Another possible explanation for the stress tolerance is that PARPs regulate stress signaling pathways at the transcriptional level, either by direct control or indirectly via abscisic acid signaling [31].

3.3. PARPs and biotic stress response

PARylation has also been implicated in plant biotic stress responses, i.e., in its defense against pathogens. Cellular levels of PAR increased upon Pseudomonas infection and pathogen-dependent changes in the PARylation of acceptor proteins were also demonstrated [32]. PARP activation did not lead to significant depletion of NAD+ pools after activation of the PARP enzyme [32]. Recent data has shown that treatment of Arabidopsis seedlings with the microbe-associated molecular pattern (MAMP) peptide flg22, which triggers plant immune reactions, induced AtPARP2 activity in vivo without an increase in the AtPARP2 protein amount [21]. On the other hand, treatment of Arabidopsis with PARP inhibitors disrupted basal defense responses to MAMPs such as flg22 or elf18. After such treatment the early immune responses, such as accumulation of reactive oxygen species (ROS in the so-called “ROS burst”) and induction of early-response marker genes still occurred. However, later responses, induced cell wall reinforcement with callose and lignin, phenylpropanoid pigment accumulation, and phenylalanine ammonia lyase activity, were compromised [33]. Flg22 elicited dramatic and much more severe seedling growth inhibition in the presence of a PARP inhibitor suggesting that some aspects of the normal defense responses became toxic in the absence of PARP activity. Studies on parp mutants in Arabidopsis have demonstrated that parp1 parp2 double mutant showed reduced expression of MAMP-induced genes and enhanced susceptibility to virulent Pseudomonas infections [21]. Together these data suggest that PARylation plays significant and specific roles in biotic stress responses, however, the molecular mechanisms are still largely unknown.

3.4. PARGs

PARylation is a reversible PTM and the removal of the covalently attached PAR from acceptor proteins is mediated by PARG. PARG activity does not restore the NAD+ pool, but increases cellular level of PAR. In addition, PARG activity releases free acceptor proteins for further PARylation by PARPs. Thus, PARG activity can either counteract or further enhance PARP activation depending on the cellular context.

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The animal genomes sequenced thus far contain only one PARG gene and the protein exists in three different isoforms [5]. Knocking out the only PARG gene is lethal in mouse and Drosophila [34, 35]. Arabidopsis has two PARG genes in tandem configuration (At2g31870, PARG1 and At2g31865, PARG2). The products of both genes contain PARG catalytic domains and share 52 % amino acid identity and 66 % similarity. Both proteins are localized to the nucleus. The presence of two PARGs and viability of the corresponding Arabidopsis single mutants provide unique possibilities for analysis of PARylation in this model organism. The two Arabidopsis alleles of parg1 mutant, tej and aggie2, accumulate high amounts of PAR polymer and are altered in circadian rhythms and pathogen defense gene expression, respectively [21, 36], suggesting that PARG1 activity is involved in regulating these processes. The aggie2 mutant showed enhanced expression of defense genes upon treatment with MAMPs which suggest that PARG1 negatively regulates expression of MAMP-induced genes. The tej mutant was isolated in a genetic screen for altered circadian period length and is characterized by early flowering and alteration of the clock-controlled transcription of genes.

Like PARPs, plant PARGs have diverse functions in both abiotic and biotic stress responses. Despite that PARG1 expression was only found to be significantly induced in response to methyl viologen [30], analysis of parg1 mutant has shown that PARG1 is required for tolerance to drought, osmotic and oxidative stress responses in Arabidopsis [37]. PARG2 expression is up-regulated in response to methyl viologen, increased salinity, high light and drought [30]. In addition, PARG2 mRNA levels were significantly increased in response to both virulent and avirulent Pseudomonas strains, treatment with MAMPs [32], and infection with the necrotrophic fungus Botrytis cinerea [33]. Arabidopsis parg1 and parg2 single mutants did not show increased susceptibility to virulent and avirulent Pseudomonas, but an increase in susceptibility to Botrytis cineria was observed [33], suggesting a possible link between PARG and PCD. Interestingly, Feng et al. [21] have recently shown that Arabidopsis PARG2 does not possess PARG activity partially due to mutation of Gly to Leu in the conserved GGG-X7-QEE motif, whereas PARG1 is the functional PARG in vitro and in vivo. How this recent finding relates to the earlier reports on the role of PARG2 in stress responses remains to be addressed.

3.5. PARylation and development

Plant PARPs and PARylation have been implicated in different developmental processes such as flowering [36] and tracheary element differentiation [38]. Treatment with the PARP inhibitor 3AB inhibited tracheary element differentiation in pea roots and artichoke tubers [38]. AtPARP1 and AtPARP2 gene expression and PARP enzyme activity increased during the exponential growth phase of Arabidopsis cultured cells, which was associated with an increase in cellular glutathione pools suggesting a link between PARP expression and activity, redox homeostasis and regulation of the cell cycle [39].

Treatment of Arabidopsis with low concentration of PARP inhibitor for a long period (7 days) resulted in growth enhancement [40, 41]. However, at earlier time points (1-2 days) PARP inhibition reduced the growth of seedlings. Increased growth was the result of

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enhanced cell number related to shortening of cell division cycle which led to more divisions and more cells [41]. Gene expression profiling revealed that a small subset of genes responded at multiple time points to PARP inhibition. Deregulated genes often had circadian expression pattern and encoded proteins interacting with the cell cycle and the proteasome [41].

To summarize, functional PARPs and PARGs are necessary for proper plant development and stress responses. Recent data suggest that modulation of PARylation in plants might be used for creating resistant crop species which will survive in changing environment.

4. The SRO proteins

The plant-specific PARP-like domain containing SRO protein family represents a separate group of proteins with six members in Arabidopsis [15] (Fig. 3). The domain architecture of SROs is conserved: in addition to the PARP-like domain, all members of the SRO family contain an RST (RCD1-SRO-TAF4) domain at the C-terminus of the protein. The Group I SROs have additionally a WWE domain in the N-terminal part (Fig. 3). The SROs are considered to lack PARP [15, 42] or mART activity [43] since the amino acids required for catalysis are not conserved in these proteins. However, recently an exception was discovered when PARP activity was demonstrated in a cultivar-specific allele of wheat SRO protein Ta-sro1 [44], as discussed later in more detail.

4.1. The WWE domain

The Group I SROs are the only WWE domain-containing protein family in plants. The WWE domain, named by three ubiquitously conserved aminoacid residues (Trp, Trp and Glu), was originally described in several eukaryotic multidomain proteins in combination with either E3 ligase or PARP (PARP-like) domains and was originally predicted to be involved in protein-protein interactions [45], but has more recently been shown to bind PAR.

Properties of the WWE domain have not been addressed in plants and insights into its structure and functions come from the studies of the animal homologs. These include the RING-type E3 ubiquitin ligase RNF146 that acts in the Wnt signaling [46]. It has been shown that the WWE domain of RNF146 binds to iso-ADP-ribose, a structural unit of the PAR polymer. PAR-binding motif of the RNF146 WWE domain was defined [47, 48] and later the crystal structure of the domain was determined [48]. The domain was shown to form a half of a beta-barrel with a positively charged PAR-binding pocket on one of its ends. Comparison of the ligand-bound and the unligated structures of WWE showed that binding to PAR induced large conformational changes in the domain [49]. As was apparent from the structure, the conserved Trp and Glu residues were not involved in the PAR binding, but rather stabilized the barrel. A recent study demonstrated that the binding of RNF146 to PAR is mediated not by the WWE domain alone, but by a combination of the WWE and the RING domains of this protein. Moreover, binding to PAR led to structural changes in the enzyme that induced the RING domain catalytic activity [49]. In vitro assays have revealed PAR-binding properties in a number of other WWE-domain proteins [47, 48]. In plants PAR binding has been demonstrated in vitro

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for the Arabidopsis SRO protein RCD1 (Vainonen JP et al, manuscript in preparation). Thus, it might be that binding PAR is a universal feature of all WWE domains [48]. Whether it provides allosteric regulation of plant SROs’ functions remains to be determined.

4.2. The RST domain

In addition to the PARP-like domain, all SRO proteins contain an RST domain at their C-terminus [15]. This domain is involved in protein-protein interactions and can only be found in plants. In addition to SROs, only two other Arabidopsis proteins, TAF4 (TBP-associated factor 4) and its homolog TAF4b, possess the RST domain. TAF4 proteins are components of the RNA Polymerase II transcription initiation complex TFIID that binds to the promoters of transcribed genes. Studies on metazoan TAF4 have revealed their dual binding function. On one hand, they are forming a part of the core transcriptional apparatus through a DNA-binding C-terminal histone-fold domain. On the other hand, they interact with specific regulators of transcription via their N-terminal domains (ETO or CRI) [50]. It is noteworthy that the plant TAF4 proteins do not contain an ETO domain, but have an RST domain in the same relative position where the animal TAF4 proteins have the ETO domain.

Systematic in vitro studies have revealed numerous protein partners of SROs, most of which belong to various families of transcription factors [15, 51, 52]. The wide range of protein partners may be based on the molecular properties of the RST domain that contains large spans of intrinsically disordered amino acid sequences. Thus, RST does not form a defined tertiary structure in solution, but rather exists in molten state [53]. Interestingly, in vitro thermodynamic studies and in silico simulations of the binding of Arabidopsis RCD1 to transcription factors have demonstrated that the interaction does not lead to folding of the proteins into a globule, but keeps them in a “fuzzy” state [53]. Many transcription factors interacting with RCD1 also have intrinsically disordered regions. Such properties were proposed to be crucial for interaction of multiple signaling pathways in the so-called signaling hubs [54]. Binding of SRO to transcription factors was shown to occur in one-to-one stoichiometry [53]. This suggests competitive complex formation between one SRO protein and one of many transcription factors or between one transcription factor and one of many different SROs. Given the diverse physiological roles of the transcription factors involved (see below), this might serve the molecular basis for the interaction of multiple signaling pathways.

4.3. Physiological and molecular functions of SROs

Of the two PARP-domain containing protein families in plants, the PARPs and the SROs, there are more published results for the involvement of the SROs in various physiological processes than there are for the PARPs. Thus, more is known about the plant processes where the PAR binding (by the WWE-domain) PARP-like-domain containing proteins are involved than is known about the mechanisms, regulation and targets of PARylation and the role of PARPs therein. However, there is still a large gap in the understanding of how the role and function of the SROs is connected to PARylation and how these processes link and act together. Most of

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the research conducted to date with the SROs has been focused on the members of the Group I [15, 51, 52]. In Arabidopsis the Group I of SROs is represented by two proteins, RCD1 (At1g32230) and SRO1 (At2g35510). In other species it is not always possible to distinguish between RCD1 and SRO1, therefore the proteins are named “SRO1s”. In Selaginella there is only one SRO1 protein of Group I; in other species there are two or more: for example in rice and Brachypodium there are five different SRO1 proteins.

4.4. Numerous phenotypes of Arabidopsis rcd1 mutant

The Arabidopsis RCD1 appears to be an important signaling hub integrating with developmental, hormonal and stress signaling pathways. Loss-of-function mutation in Arabidopsis RCD1 leads to a pleiotropic phenotype characterized by altered plant development and stress reactions. Developmental phenotypes include inhibited elongation of shoots, aberrant growth of roots and aerial parts such as altered leaf shape, increased branching and early flowering. Among the altered stress responses of rcd1 are its increased tolerance to high sugar concentrations [55] and to ultraviolet irradiation [56, 57] and decreased tolerance to salt stress [58]. Many hormonal signaling pathways, including those controlled by the plant hormones ethylene, abscisic acid, jasmonic acid [55] and salicylic acid [59] were shown to be altered in rcd1. Thus, in Arabidopsis the RCD1 protein seems to be involved in all these separate processes.

The growth phenotypes of rcd1 are characteristic of the so-called stress-induced morphogenic response (SIMR, [60]). The SIMR is orchestrated through signaling by ROS and a number of plant hormones, including salicylic acid, auxin and ethylene [60]. During pathogen infections this developmental strategy allows a plant to allocate energy resources to defense rather than growth. Therefore, the rcd1 mutant demonstrates the symptoms of constitutive defense response [60, 61]. Introduction of rcd1 mutation into the snc1 mutant background, which also displays continuous immune response, enhanced the growth arrest phenotype without increasing the actual resistance to pathogens of the double mutant [62]. This dissection of defensive and developmental branches of immune signaling placed RCD1 in the latter, pointing at its involvement in SIMR.

Other prominent phenotypes of rcd1 most probably connected to its constitutive defense status are its altered sensitivity to ROS. One of the first alleles of rcd1 was isolated due to increased cell death of this mutant under the treatment of plants with ozone, a gas that enters intracellular space in the leaf tissues leading to production of ROS in the cell wall [63]. Such ROS formation mimics natural ROS burst occurring during plant immune responses. The ozone-related phenotype gave RCD1 its name. Interestingly, the hypersensitivity of the mutant to extracellular ROS coincides with significantly increased tolerance of rcd1 to the treatments that stimulate formation of ROS in the chloroplasts, including increased light intensities and application of the herbicide methyl viologen [56, 59, 64].

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Altogether these numerous phenotypes suggest that the wild type RCD1 protein, which with its N-terminal end (via the WWE-PARP module) binds PAR and at its C-terminal end (via the RST domain) interacts with specific transcription factors, is a co-regulator in several developmental and inducible processes where protein PARylation apparently plays a role. All the known rcd1 alleles are lacking the C-terminal RST domain. Accordingly all the phenotypes in the rcd1 mutants are a result of disrupted interaction with the interacting transcription factors or other proteins. However, the significance and role of the PAR-binding N terminus of RCD1 is not known.

4.5. Interaction partners of RCD1

Many of the observed rcd1 phenotypes may be explained by protein-protein interaction of RCD1 with numerous transcription factors. One of these factors, ANAC013, was shown to localize to endoplasmic reticulum and to migrate to nucleus upon its proteolytic cleavage [65]. In the nucleus ANAC13 induces the expression of a number of genes implicated in mitochondrial redox metabolism [65]. The rcd1 mutant is characterized by up-regulation of the above-mentioned genes under unstressed conditions [59], which suggests that binding to RCD1 is involved in the regulation of the activity of ANAC013 [65].

Another transcription factor interacting with RCD1 is DREB2A, one of the key transcriptional regulators involved in plant response to heat stress, drought response and senescence. In vitro deletion analyses revealed the presence of a motif FDXXELLXXLN (RIM) in the DREB2 transcription factors that was shown to interact with the RCD1 RST-domain [66]. Removal of the RIM-domain from DREB2A abolished the interaction of DREB2A with RCD1 in yeast-two-hybrid system. An alternative splice variant of DREB2A lacking the RIM motif, and thus unable to interact with RCD1, was shown to accumulate during plant senescence and heat stress. It was hypothesized that RCD1 negatively affects DREB2A activity [66].

Expression of several genes encoding chloroplast-localized antioxidant enzymes was shown to be impaired in rcd1. Since these genes were responsive to the transcription factor Rap2.4a, the interaction between this protein and RCD1 was suggested. Indeed, the two proteins did interact in yeast [67]. Rap2.4a has a motif FDXXEeaXXLa that resembles the RIM of DREB2A. Whether the interaction with RCD1 is positive or negative might depend on the age of the plant [67].

Similarly, tolerance to ultraviolet light and up-regulation of ultraviolet-responsive genes in rcd1 could be explained by the interaction of RCD1 with a B-box zinc finger transcription factor STO/BBX24 involved in light signaling [68]. Altered cell death in rcd1 could arise from the altered function of transcription factors WRKY70 and SGT1b, as was shown by extensive genetic studies [59].

RCD1 was also found to interact with the C-terminal cytoplasmic tail of the plasma membrane-localized Na+/H+ antiporter SOS1 in yeast-two-hybrid assays. Under non-stressed condition RCD1 was localized to the nucleus, however, under high salt or H2O2 treatment RCD1

14

was present also outside of the nucleus close to the cell periphery [58]. Such nuclear-cytoplasm stress-induced redistribution suggests a role of RCD1 also outside the nucleus.

4.6. Arabidopsis rcd1 sro1 double mutant

In the only available allele of the sro1 mutant the T-DNA insertion at end of the SRO1 gene results in expression of truncated protein that lacks the RST domain. In contrast to rcd1, this sro1 mutant is only slightly affected in development and stress responses. Most of the developmental phenotypes tested are reverted in sro1 as compared to rcd1 and are also present in very mild form. For example, while rcd1 is characterized by early flowering, in sro1 it is slightly retarded. In contrast to rcd1, sro1 has slightly increased tolerance to apoplastic ROS and to salt stress [69]. Recently it was shown that sro1 mutant was hypersensitive to HgCl2 [70].

However, the rcd1 sro1 double mutant demonstrated severe developmental defects [51, 69]. These defects are manifested already at the initial stages of embryogenesis with some individuals being arrested as early as at the globular stage and others displayed various abnormal phenotypes at the heart stage. Rare double mutant individuals that proceed to germination have extremely shortened hypocotyls [71]. These double mutants were only able to survive in sugar-containing sterile medium; however, their growth was extremely stunted [51, 69]. Interestingly, the rcd1-/- sro1+/- progeny of the rcd1 to sro1 cross also demonstrated stunted growth, although less pronounced than in the homozygous double mutant [51]. The described abnormalities were suggested to be caused by the compromised division-competent status of meristematic cells and premature exit of cells from the cell cycle. As no significant changes were observed in the double mutant in relation to the proteins involved in cell fate determination or auxin transport, it was concluded that the major cause of the striking phenotype is the unbalanced redox metabolism leading to pronounced SIMR [60, 69]. The high similarity of RCD1 and SRO1 proteins together with pleiotropic phenotypes of rcd1, insignificant and mild phenotypes of sro1 and severe developmental defects of the double rcd1 sro1 mutant, brought about the concept of unequal redundancy of these two proteins [51].

4.7. Other SROs

Certain members of the SRO Group I have been shown to play similarly important roles in plant species other than the best-studied Arabidopsis. For example, transcriptomic analyses in rice have shown that one of its SRO genes named OsSRO1c is highly inducible under different stress treatments [42]. Similarly to Arabidopsis rcd1, ossro1c showed increased tolerance to chloroplast ROS generated by methyl viologen; however, contrary to rcd1, it was also tolerant to apoplastic ROS introduced by plant growth in presence of H2O2 and hypersensitive to cold treatment. Yeast-two-hybrid assays revealed interaction of 29 transcription factors from fifteen different families with OsSRO1c [52]. All those interactions (except for OsDREB2B) required the PARP-like domain of OsSRO1c in addition to the RST domain [52].

15

A specific allele of wheat SRO1 protein named Ta-sro1 conferred higher resistance to salt and to ultraviolet irradiation to a wheat cultivar SR3. In that particular cultivar Ta-sro1 gene was overexpressed, which correlated with elevated endogenous levels of ROS [44]. In contrast to the homologous protein Ta-SRO1 form the parental cultivar and to all other plant SROs characterized to date, Ta-sro1 was reported to possess PARP activity in vitro and in vivo. The molecular reason for this might be several amino acid replacements that lead to efficient binding of NAD+ by the PARP-like domain. Heterologous overexpression of Ta-sro1 in other wheat cultivars or in Arabidopsis resulted in altered expression and activity of enzymes implicated in ROS production and scavenging [44].

The role of PARP-like domains in the functions of the proteins described above is unclear. Several hypotheses explaining the function of SROs in transcriptional regulation have been proposed [72]. The proteins could interact with PARylated components of chromatin and transcriptional machinery via the WWE-PARP modules and recruit specific transcription factors to their target sequences via the RST domain. In such PAR-protein-DNA complex the RST domain of SRO could be replaced with that of TAF4 to initiate transcription. Alternatively, the SRO RST domain could compete with that of TAF4 for binding with transcription factors, thereby inhibiting the action of the latter. These two scenarios would correspond to positive and negative effect of the SRO protein on its interacting transcription factors, accordingly.

5. Concluding remarks and open questions

PARPs and PARGs modify acceptor proteins by addition or removal of PAR. Over the past two decades it has been shown that PARP family and PARylation are involved in a variety of biological functions in plants, including DNA repair, transcription and cell death. However, the molecular mechanisms and signaling pathways which activate and regulate PARPs and PARGs remain to be addressed.

Despite the information about structure and function of PARP and PARGs, little is known about acceptors protein of poly(ADP-ribose) and proteins interacting with PAR. No PARylated proteins except histones and PARPs have been identified in plants. Identification of new poly(ADP-ribose) acceptor proteins will help in understanding the regulatory function of PARylation in plant development and stress responses. Whether PAR cleaved from its acceptor proteins through PARG activity acts as a signal in plants similarly to animals is yet to be determined.

In spite of conservation of the PARP-like domain, the SRO proteins are predicted to lack poly- or mono-ADP-ribose transferase activity [15, 43]. Nevertheless RCD1 and SRO1 play important roles in plant stress and development responses as follows from the mutant studies. Interaction with a large number of stress-responsive transcription factors and the presence of the WWE domain shown to interact with PAR suggest a role for RCD1 and SRO1 in transcriptional regulation in PAR-dependent manner. RCD1 and SRO1 may function as scaffold proteins which can positively or negatively regulate transcription based on what complexes are

16

formed. Future research in regulation of RCD1 and identification of protein complexes formed by RCD1 and SRO1 will provide valuable information on signaling pathways acting in concert to achieve rapid and appropriate response to the external stimuli.

17

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Figure legends

Figure 1. Maximum likelihood phylogenetic tree of the PARP domain containing proteins in plants. Protein models with PARP domains were retrieved from 29 plant and algal species listed in Supplementary table S1. The alignment and the phylogenetic tree were created using PASTA. Algae Volvox carteri and Chlamydomonas reinhardtii contained only one protein model with PARP domain. Together a PARP protein model from Physcomitrella patens these genes form the outgroup for the main clades of plant PARPs and SROs. Names for PARP clades come from the Arabidopsis thaliana genes included in each group and naming of the SROs corresponds the naming in Jaspers et al [15]. The branches of the tree shown here are shown in more detail with the individual proteins in Supplementary figures S1-S6.

Figure 2. Maximum likelihood phylogenetic tree of the PARG domain containing proteins in plants. The alignment and the phylogenetic tree were produced with PASTA from full protein models annotated from 29 plant and algal species listed in Supplementary table S1. Human PARG was used as the outgroup. Only land plant species contained PARG domains.

Figure 3. Conserved protein domains in human (A) and Arabidopsis (B) PARPs and PARP-like proteins. Domain structure was determined using Pfam database.

PARP reg – PARP regulatory domain; PARP cat – PARP catalytic domain; WGR – putative PARP nucleic acid binding domain; RST domain (RCD1-SRO-TAF4) – plant specific protein-protein interaction domain; WWE domain – poly(ADP-ribose) binding domain; BRCT domain (BRCA1 carboxy-terminal domain) is found within many DNA damage repair and cell cycle checkpoint proteins; ANK – ankyrin repeat domain – mediates protein-protein interactions; SAM (sterile alpha motif) – is found in signaling and nuclear proteins; VIT (vault protein inter-alpha-trypsin) and VWA (von Willebrand type A) domains – mediate protein-protein interactions; ZF - zinc-finger domains; SAP – putative DNA-binding domain; UIM - ubiquitin interaction motif; Macro domain – a domain that can serve as the ADP-ribose or the O-acetyl-ADPribose binding module; PADPR1 – domain of unknown function.

0.5

SROIb

SROIa

SROIc

Amborella trichopoda SRO2a

Spirodela polyrhiza SRO2a

SROIIa

SROIIb

PARP1

PARP3

Homo sapiens PARP1

Homo sapiens PARP2

Homo sapiens PARP3

PARP2

Physcomitrella patens PARP

Volvox carteri PARP

Chlamydomonas reinhardtii PARP

Figure 1.Maximum likelihood phylogenetic tree of the PARP domain containing proteins in plants. Protein models with PARP domains were retrieved from 29 plant and algal species listed in Supplementary table S1. The alignment and the phylogenetic tree were created using PASTA. Algae Volvox carteri and Chlamydomonas reinhardtii contained only one protein model with PARP domain. Together with a PARP protein model from Physcomitrella patens these genes form the outgroup for the main clades of plant PARPs and SROs. Names for PARP clades come from the Arabidopsis thaliana genes included in each group and naming of the SROs corresponds the naming in Jaspers et al. [15]. The branches of the tree shown here are shown in more detailwith the individual proteins in Supplementary figures S1-S6.

Selaginella moellendorffii PARG1aSelaginella moellendorffii PARG1b

Physcomitrella patens PARG1aPhyscomitrella patens PARG1b

Oryza sativa PARG1aBrachypodium distanchyon PARG1a

Brachypodium distanchyon PARG1bBrachypodium distanchyon PARG1c

Brachypodium distanchyon PARG1dHordeum vulgare PARG1a

Zea mays PARG1aSorghum bicolor PARG1a

Spirodela polyrhiza PARG1aAmborella trichopoda PARG1a

Aquilegia coerulia PARG1aSolanum melongena PARG1a

Solanum tuberosum PARG1aSolanum lycopersicum PARG1a

Solanum melongena PARG1bSolanum tuberosum PARG1bSolanum lycopersicum PARG1b

Capsella rubella PARG1aArabidopsis thaliana PARG1

Arabidopsis lyrata PARG1aCapsella rubella PARG1b

Arabidopsis thaliana PARG2Arabidopsis lyrata PARG1b

Capsella rubella PARG1c

Capsella rubella PARG1dArabidopsis lyrata PARG1c

Vitis vinifera PARG1aEucalyptus grandis PARG1a

Prunus persica PARG1aMedicago truncatula PARG1a

Glycine max PARG1aGlycine max PARG1b

Cucumis sativus PARG1aBetula pendula PARG1a

Betula pendula PARG1bTheobroma cacao PARG1aPopulus trichocarpa PARG1aPopulus trichocarpa PARG1b

Homo sapiens PARG

0.3

Figure 2. Maximum likelihood phylogenetic tree of the PARG domain containing proteins in plants. The alignment and the phylogenetic tree were produced with PASTA from full protein models annotated from 29 plant and algal species listed in Supplementary table S1. Human PARG was used as the outgroup. Only land plant species contained PARG domains.

COOHNH2PARP1

COOHNH2PARP2

COOHNH2PARP3

COOHNH2PARP4

COOHNH2PARP5a

COOHNH2PARP5b

COOHNH2PARP15

COOHNH2PARP14

COOHNH2PARP9

COOHNH2PARP10

COOHNH2PARP11

COOHNH2PARP12

COOHNH2PARP13

COOHNH 2PARP7

COOHNH2PARP16

COOHNH2PARP8

COOHNH2PARP6

COOHNH2

NH2COOH

NH2 COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOHPARP1

PARP2

PARP3

RCD1

SRO1

SRO2

SRO3

SRO4

SRO5

PARPcat

WWE

RST

BRCT

PARPreg

Zn finger

WGR

PADR1

SAM

ANK

Macro

UIM

VIT VWA

SAP

Figure 3. Conserved protein domains in human (A) and Arabidopsis (B) PARPs and PARP-like proteins. Domain structure was determined using Pfam database. PARP reg – PARP regulatory domain; PARP cat – PARP catalytic domain; WGR – putative PARP nucleic acid binding domain; RST domain (RCD1-SRO-TAF4) – plant specific protein-protein interactiondomain; WWE domain – poly(ADP-ribose) binding domain; BRCT domain (BRCA1 carboxy-terminal domain) is found within many DNA damage repair and cell cycle checkpoint proteins; ANK – ankyrin repeat domain – mediates protein-protein interactions; SAM (sterile alpha motif) – is found in signaling and nuclear proteins; VIT (vault protein inter-alpha-trypsin) and VWA (von Willebrand type A) domains – mediate protein-protein interactions; ZF - zinc-finger domains; SAP – putative DNA-binding domain; UIM - ubiquitin interaction motif; Macro domain – a domain that can serve as the ADP-ribose or the O-acetyl-ADPribose binding module; PADPR1 – domain of unknown function.

0.5

Sorghum bicolor PARP1a

Physcomitrella patens PARP1a

Populus trichocarpa PARP1a

Cucumis sativus PARP1a


SROIIb

Solanum tuberosum PARP1a

Hordeum vulgare PARP1a

Betula pendula PARP1a

Zea mays PARP1a

Solanum lycopersicum PARP1a

Glycine max PARP1a

Arabidopsis thaliana PARP1


Theobroma cacao PARP1a

Spirodela polyrhiza PARP1a

Homo sapiens PARP3

Aquilegia coerulea PARP1a

Oryza sativa PARP1a

Capsella rubella PARP1a


Medicago truncatula PARP1a

Arabidopsis lyrata PARP1a

SROIIa

SROIa

Prunus persica PARP1a

Amborella trichopoda PARP1a


Glycine max PARP1b

Homo sapiens PARP2

Theobroma cacao PARP1b

SROIc

Vitis vinifera PARP1a

PARP3

Volvox carteri PARP

Homo sapiens PARP1

PARP2

Solanum melongena PARP1a

Brachybodium distachyon PARP1a

SROIb

Eucalyptus grandis PARP1a

Selaginella moellendorffii PARP1a

Supplementary Figure S1. The PARP1 clade in phylogenetic tree shown in Figure 1

0.5



Selaginella moellendorffii PARP2b

Sorghum bicolor PARP2b



Spirodela polyrhiza PARP2aTheobroma cacao PARP2a

SROIIb


Amborella trichopoda PARP2b


Zea mays PARP2a

SROIb

Oryza sativa PARP2b


Homo sapiens PARP2

Solanum lycopersicum PARP2b

Homo sapiens PARP1

Populus trichocarpa PARP2b





Aquilegia coerulea PARP2c




Homo sapiens PARP3

Glycine max PARP2a


Solanum tuberosum PARP2b

Glycine max PARP2bMedicago truncatula PARP2a

Aquilegia coerulea PARP2b

PARP3

Amborella trichopoda PARP2a


Medicago truncatula PARP2b

SROIa

Oryza sativa PARP2a


PARP1


Volvox carteri PARP




SROIc

SROIIa


Supplementary Figure S2. The PARP2 clade in the phylogenetic tree shown in Figure 1

0.5

Spirodela polyrhiza PARP3a



Glycine max PARP3a

Zea mays PARP3a

Homo sapiens PARP2


Volvox carteri PARP

Solanum melongena PARP3b


SROIc



Homo sapiens PARP3





Solanum_tuberosum_PARP3b

PARP1

SROIb

Populus trichocarpa PARP3b

SROIIa

Solanum lycopersicum PARP3b

SROIa




PARP2


Oryza sativa PARP3a



Medicago truncatula PARP3a


SROIIb

Theobroma cacao PARP3a

Homo sapiens PARP1


Glycine max PARP3b





Supplementary Figure S3. The PARP3 clade in the phylogenetic tree shown in Figure 1.

0.5

Betula pendula SRO1b

Hordeum vulgare SRO1b

Solanum melongena SRO1a


Solanum lycopersicum SRO1a

Cucumis sativus SRO1a

Solanum tuberosum SRO1a

Zea mays SRO1b

Betula pendula SRO1a

Solanum tuberosum SRO1b

Capsella rubella SRO1a

Arabidopsis lyrata SRO1b

Aquilegia coerulea SRO1b

SROIb


Populus trichocarpa SRO1aPopulus trichocarpa SRO1b

Oryza sativa SRO1b

Solanum lycopersicum SRO1c

Brachybodium distachyon SRO1a


Aquilegia coerulea SRO1a

Theobroma cacao SRO1a

Sorghum bicolor SRO1b

Medicago truncatula SRO1b

Homo sapiens PARP1

PARP1

Solanum tuberosum SRO1c


Capsella rubella SRO1b

SROIIb

Glycine max SRO1b

Solanum melongena SRO1b

Vitis vinifera SRO1a

Amborella trichopoda SRO1b

PARP3

Homo sapiens PARP2

Arabidopsis thaliana RCD1

Glycine max SRO1a

Prunus persica SRO1a

Solanum lycopersicum SRO1b

Medicago truncatula SRO1a

SROIIa

Zea mays SRO1c

Vitis vinifera SRO1b

Arabidopsis thaliana SRO1

Eucalyptus grandis SRO1a

PARP2

Brachybodium distachyon SRO1b

Oryza sativa SRO1a

Hordeum vulgare SRO1a

SROIc


Volvox carteri PARPChlamydomonas reinhardtii PARP

Medicago truncatula SRO1c

Homo sapiens PARP3

Sorghum bicolor SRO1a

Cucumis sativus SRO1b

Zea mays SRO1a

Arabidopsis lyrata SRO1a

Supplemenatry Figure S4. SROIa clade in the phylogenetic tree shown in Figure 1.

0.5

Selaginella moellendorffii SRO1a

Sorghum bicolor SRO1e


Physcomitrella patens SRO1c

Brachybodium distachyon SRO1c

Solanum tuberosum SRO1e

Amborella trichopoda SRO1c

Solanum melongena SRO1c

Betula pendula SRO1c

Solanum lycopersicum SRO1d

Zea mays SRO1e

Oryza sativa SRO1c

SROIIb

Solanum tuberosum SRO1d

PARP1


SROIa

Prunus persica SRO1bSolanum lycopersicum SRO1i

Hordeum vulgare SRO1d

Sorghum bicolor SRO1d

Brachybodium distachyon SRO1e


Solanum lycopersicum SRO1e

Theobroma cacao SRO1c

Volvox carteri PARP

Oryza sativa SRO1d

Solanum tuberosum SRO1f

Homo sapiens PARP3Homo sapiens PARP2


Cucumis sativus SRO1c

Zea mays SRO1d

Eucalyptus grandis SRO1b

Homo sapiens PARP1

Sorghum bicolor SRO1c

Physcomitrella patens SRO1a

Zea mays SRO1f

Oryza sativa SRO1e

PARP2

Solanum lycopersicum SRO1h

Solanum lycopersicum SRO1gSolanum lycopersicum SRO1f

Theobroma cacao SRO1b

Hordeum vulgare SRO1c

SROIIa

Populus trichocarpa SRO1c

PARP3

Brachybodium distachyon SRO1d

Physcomitrella patens SRO1b

Supplementary Figure S5.SROIb and SROIc clades in the phylogenetic tree shown in Figure 1.

0.5

Medicago truncatula SRO2b

Populus trichocarpa SRO2b

Populus trichocarpa SRO2d

Solanum tuberosum SRO2b


Homo sapiens PARP2

Medicago truncatula SRO2a

Betula pendula SRO2a

PARP1

Arabidopsis lyrata SRO2b


Solanum tuberosum SRO2c

Homo sapiens PARP1

Eucalyptus grandis SRO2c

Arabidopsis lyrata SRO2a

Solanum lycopersicum SRO2c

PARP3

SROIc

Homo sapiens PARP3

Volvox carteri PARP

Capsella rubella SRO2a

SROIb

Solanum melongena SRO2b



Prunus persica SRO2b

Aquilegia coerulea SRO2a


Capsella rubella SRO2b

Theobroma cacao SRO2b

PARP2

Arabidopsis lyrata SRO2d

Eucalyptus grandis SRO2a

Arabidopsis lyrata SRO2c

Solanum lycopersicum SRO2bSolanum tuberosum SRO2a

Theobroma cacao SRO2a

Prunus persica SRO2a

Solanum lycopersicum SRO2aSolanum melongena SRO2a

SROIa

Betula pendula SRO2b

Populus trichocarpa SRO2e


Capsella rubella SRO2c

Cucumis sativus SRO2aPopulus trichocarpa SRO2f

Vitis vinifera SRO2a


Vitis vinifera SRO2b

Capsella rubella SRO2d

Populus trichocarpa SRO2a

Solanum melongena SRO2c

Glycine max SRO2a


Eucalyptus grandis SRO2b

Populus trichocarpa SRO2c

Supplementary Figure S6. SROIIa and SROIIb clades in the phylogenetic tree shown in Figure 1.

Supplementary Table S1. Plant species used in phylogenetic analyses.

Scientific name Common name Source

Amborella trichopoda Amborella Phytozome

Aquilegia coerulea Columbine Phytozome

Arabidopsis lyrata Lyrate rockcress Phytozome

Arabidopsis thaliana Thale cress Phytozome

Betula pendula Birch Helsinki

Brachybodium distachyon Purple false brome Phytozome

Capsella rubella Red shepherd's purse Phytozome

Chlamydomonas reinhardtii Phytozome

Coccomyxa subellipsoidea Phytozome

Cucumis sativus Cucumber Phytozome

Eucalyptus grandis Eucalyptus Phytozome

Glycine max Soybean Phytozome

Hordeum vulgare Barley Gramene

Medicago truncatula Barrel medic Phytozome

Oryza sativa Rice Phytozome

Ostreococcus lucimarinus Phytozome

Physcomitrella patens Phytozome

Populus trichocarpa Poplar Phytozome

Prunus persica Peach Phytozome

Selaginella moellendorffii Phytozome

Solanum lycopersicum Tomato Phytozome

Solanum melongena Eggplant Eggplant database

Solanum tuberosum Potato Phytozome

Sorghum bicolor Phytozome

Spirodela polyrhiza Duckweed Phytozome

Theobroma cacao Cacao Phytozome

Vitis vinifera Grapevine Phytozome

Volvox carteri Phytozome

Zea mays Maize Phytozome

Supplementary Table S2. Nomenclature of the PARPs and SROs.

Species Gene name Original gene code

Amborella trichopoda PARP1a Manual annotation

Amborella trichopoda PARP2a evm_27.model.AmTr_v1.0_scaffold00057.282

Amborella trichopoda PARP2b evm_27.model.AmTr_v1.0_scaffold00044.52

Amborella trichopoda PARP3a Manual annotation (partial model)

Amborella trichopoda SRO1a evm_27.model.AmTr_v1.0_scaffold00048.66

Amborella trichopoda SRO1b evm_27.model.AmTr_v1.0_scaffold00024.23

Amborella trichopoda SRO1c evm_27.model.AmTr_v1.0_scaffold00056.145

Amborella trichopoda SRO2a evm_27.model.AmTr_v1.0_scaffold00058.201

Aquilegia coerulea PARP1a Aquca_017_00682.1

Aquilegia coerulea PARP2a Manual annotation

Aquilegia coerulea PARP2b Aquca_003_00704.1

Aquilegia coerulea PARP2c Aquca_003_00705.1

Aquilegia coerulea PARP3a Aquca_072_00152.1

Aquilegia coerulea SRO1a Aquca_005_00560.1

Aquilegia coerulea SRO1b Aquca_012_00072.1

Aquilegia coerulea SRO2a Aquca_025_00341.1

Arabidopsis lyrata PARP1a 902162



Arabidopsis lyrata SRO1a 473413

Arabidopsis lyrata SRO1b 482511

Arabidopsis lyrata SRO2a gi|297845414|ref|XP_002890588.1|

Arabidopsis lyrata SRO2b gi|297841797|ref|XP_002888780.1|

Arabidopsis lyrata SRO2c ARALYDRAFT_323380

Arabidopsis lyrata SRO2d Manual annotation

Arabidopsis thaliana PARP1 AT2G31320.1



Arabidopsis thaliana RCD1 AT1G32230.1

Arabidopsis thaliana SRO1 AT2G35510.1

Arabidopsis thaliana SRO2 AT1G23550


Arabidopsis thaliana SRO4 AT3G47720


Betula pendula PARP1a evm.model.Contig50.91

Betula pendula PARP2a evm.model.Contig597.32

Betula pendula PARP3a 631695C3B67CD3876772D87DFDD3FA7D

Betula pendula SRO1a E64043F855573E70F5EB5F1532EDE5AB

Betula pendula SRO1b evm.model.Contig428.33

Betula pendula SRO1c evm.model.Contig828.12

Betula pendula SRO2a evm.model.Contig38.19

Betula pendula SRO2b evm.model.Contig890.9

Brachybodium distachyon PARP1a Bradi1g52530.2.p



Brachybodium distachyon SRO1a Bradi3g34250.1.p

Brachybodium distachyon SRO1b Bradi1g01170.2.p

Brachybodium distachyon SRO1c Bradi1g69140.1.p

Brachybodium distachyon SRO1d Bradi5g25800.1.p

Brachybodium distachyon SRO1e Bradi1g63360.5.p

Capsella rubella PARP1a Carubv10022570m



Capsella rubella SRO1a Carubv10008689m

Capsella rubella SRO1b Carubv10022903m

Capsella rubella SRO2a CARUB_v10009796mg

Capsella rubella SRO2b Carubv10020702m

Capsella rubella SRO2c CARUB_v10019284mg

Capsella rubella SRO2d Carubv10027743m

Chlamydomonas reinhardtii PARP Cre17.g738550.t1.1

Cucumis sativus PARP1a Cucsa.053430.1



Cucumis sativus SRO1a Cucsa.322070.1

Cucumis sativus SRO1b Cucsa.312850.1

Cucumis sativus SRO1c Cucsa.160950.1

Cucumis sativus SRO2a Cucsa.098420.1

Eucalyptus grandis PARP1a Eucgr.K03285.1

Eucalyptus grandis PARP2a Eucgr.H01106.1

Eucalyptus grandis PARP3a Eucgr.J00484.1

Eucalyptus grandis SRO1a Eucgr.E00230.1

Eucalyptus grandis SRO1b Eucgr.F00472.1

Eucalyptus grandis SRO2a Eucgr.B00305.1

Eucalyptus grandis SRO2b Eucgr.B00313.1

Eucalyptus grandis SRO2c Eucgr.B00314.1

Glycine max PARP1a Glyma03g31820.2

Glycine max PARP1b Glyma19g34580.1





Glycine max SRO1a Glyma01g01900.8

Glycine max SRO1b Glyma09g34000.4

Glycine max SRO2a Glyma08g12963.1

Hordeum vulgare PARP1a MLOC_5569.3



Hordeum vulgare SRO1a MLOC_44162.2

Hordeum vulgare SRO1b MLOC_66554.1

Hordeum vulgare SRO1c MLOC_56445.10

Hordeum vulgare SRO1d MLOC_68071.1

Medicago truncatula PARP1a Medtr7g096520.1


Medicago truncatula PARP2b Medtr1g088400.1


Medicago truncatula SRO1a Medtr1g112330.2

Medicago truncatula SRO1b Medtr5g029580.1

Medicago truncatula SRO1c Medtr7g011550.1

Medicago truncatula SRO2a Medtr8g088250.1

Medicago truncatula SRO2b Medtr3g077870.1

Oryza sativa PARP1a LOC_Os07g23110.1


Oryza sativa PARP2b sp|Q0JMY1|PRP2B_ORYSJ


Oryza sativa SRO1a LOC_Os10g42710.1

Oryza sativa SRO1b LOC_Os03g63770.4

Oryza sativa SRO1c LOC_Os03g12820.1

Oryza sativa SRO1d Manual annotation

Oryza sativa SRO1e LOC_Os04g57640.1

Physcomitrella patens PARP Phpat.023G076000.1.p

Physcomitrella patens PARP1a Phpat.008G064000.1.p



Physcomitrella patens SRO1a Phpat.016G023800.1.p

Physcomitrella patens SRO1b Phpat.025G004900.1.p

Physcomitrella patens SRO1c Phpat.002G059200.1.p

Populus trichocarpa PARP1a Potri.002G041300.1


Populus trichocarpa PARP2b Manual annotation


Populus trichocarpa PARP3b Potri.009G143900.1

Populus trichocarpa SRO1a Potri.003G096700.1

Populus trichocarpa SRO1b Potri.001G137200.1

Populus trichocarpa SRO1c Potri.002G112300.1

Populus trichocarpa SRO2a Potri.018G055100.1

Populus trichocarpa SRO2b Potri.006G231600.1

Populus trichocarpa SRO2c Potri.006G231100.1

Populus trichocarpa SRO2d Potri.006G231500.1

Populus trichocarpa SRO2e Potri.012G081100.1

Populus trichocarpa SRO2f Potri.015G076500.1

Prunus persica PARP1a ppa000811m



Prunus persica SRO1a ppa003072m

Prunus persica SRO1b ppa006683m

Prunus persica SRO2a ppa012602m

Prunus persica SRO2b ppa007712m

Selaginella moellendorffii PARP1a 90144


Selaginella moellendorffii PARP2b 76668


Selaginella moellendorffii SRO1a Manual annotation

Solanum lycopersicum PARP1a Solyc03g117970.2.1


Solanum lycopersicum PARP2b Solyc08g074730.1.1


Solanum lycopersicum PARP3b Solyc01g009470.1.1

Solanum lycopersicum SRO1a Solyc08g005270.2.1

Solanum lycopersicum SRO1b Solyc08g076420.2.1

Solanum lycopersicum SRO1c Solyc06g066330.2.1

Solanum lycopersicum SRO1d Solyc04g077100.2.1

Solanum lycopersicum SRO1e Solyc04g077090.1.1

Solanum lycopersicum SRO1f Solyc04g077080.1.1

Solanum lycopersicum SRO1g Solyc01g057370.1.1

Solanum lycopersicum SRO1h Solyc04g077070.1.1

Solanum lycopersicum SRO1i Solyc01g057360.1.1

Solanum lycopersicum SRO2a Solyc05g005280.2.1

Solanum lycopersicum SRO2b Solyc05g005290.2.1

Solanum lycopersicum SRO2c Solyc03g114360.2.1

Solanum melongena PARP1a Manual annotation

Solanum melongena PARP2a Sme2.5_01840.1_g00005.1

Solanum melongena PARP3a Sme2.5_01446.1_g00005.1

Solanum melongena PARP3b Sme2.5_05911.1_g00002.1

Solanum melongena SRO1a Sme2.5_00579.1_g00011.1

Solanum melongena SRO1b Sme2.5_06361.1_g00001.1

Solanum melongena SRO1c Sme2.5_00039.1_g00015.1

Solanum melongena SRO2a Sme2.5_00744.1_g00001.1

Solanum melongena SRO2b Sme2.5_02377.1_g00010.1

Solanum melongena SRO2c Sme2.5_24311.1_g00001.1

Solanum tuberosum PARP1a Manual annotation

Solanum tuberosum PARP2a PGSC0003DMP400052366

Solanum tuberosum PARP2b PGSC0003DMP400052367

Solanum tuberosum PARP3a PGSC0003DMP400013102

Solanum tuberosum PARP3b Manual annotation

Solanum tuberosum SRO1a gi|565391104|ref|XP_006361265.1|

Solanum tuberosum SRO1b PGSC0003DMP400026058

Solanum tuberosum SRO1c PGSC0003DMP400029296

Solanum tuberosum SRO1d PGSC0003DMP400008837

Solanum tuberosum SRO1e PGSC0003DMP400008838

Solanum tuberosum SRO1f PGSC0003DMP400064756

Solanum tuberosum SRO2a PGSC0003DMP400023842

Solanum tuberosum SRO2b PGSC0003DMP400023832

Solanum tuberosum SRO2c PGSC0003DMP400042580

Sorghum bicolor PARP1a Sobic.002G116300.1.p


Sorghum bicolor PARP2b Sobic.003G159200.1.p


Sorghum bicolor SRO1a Sobic.001G284100.1.p

Sorghum bicolor SRO1b Sobic.001G006700.1.p

Sorghum bicolor SRO1c Sobic.006G262500.1.p

Sorghum bicolor SRO1d Sobic.008G002500.1.p

Sorghum bicolor SRO1e Sobic.001G447100.1.p

Spirodela polyrhiza PARP1a Spipo16G0040200

Spirodela polyrhiza PARP2a Manual annotation (partial model)

Spirodela polyrhiza PARP3a Spipo0G0005500

Spirodela polyrhiza SRO1a Spipo30G0006900

Spirodela polyrhiza SRO2a Manual annotation

Theobroma cacao PARP1a Thecc1EG004107t2

Theobroma cacao PARP1b Thecc1EG004119t1



Theobroma cacao SRO1a Thecc1EG016592t4

Theobroma cacao SRO1b Thecc1EG034612t1

Theobroma cacao SRO1c Thecc1EG017921t1

Theobroma cacao SRO2a Thecc1EG010372t1

Theobroma cacao SRO2b Thecc1EG012073t1

Vitis vinifera PARP1a GSVIVT01028296001



Vitis vinifera SRO1a GSVIVT01013090001

Vitis vinifera SRO1b GSVIVT01013086001

Vitis vinifera SRO2a GSVIVT01013740001

Vitis vinifera SRO2b GSVIVT01007754001

Volvox carteri PARP Vocar20012737m

Zea mays PARP1a GRMZM5G831712_T02

Zea mays PARP2a sp|O50017|PARP2_MAIZE

Zea mays PARP3a GRMZM2G038536_T01

Zea mays SRO1a GRMZM2G145236_T02

Zea mays SRO1b GRMZM5G866843_T03

Zea mays SRO1c GRMZM2G072894_T01

Zea mays SRO1d Manual annotation

Zea mays SRO1e GRMZM2G122543_T01

Zea mays SRO1f GRMZM2G177878_T02

Homo sapiens PARP1 P09874

Homo sapiens PARP2 Q9UGN5

Homo sapiens PARP3 Q9Y6F1

Supplementary Table S3. Nomenclature of the PARGs.

Species Gene name Original gene code

Amborella trichopoda PARG1a evm_27.model.AmTr_v1.0_scaffold00066.92

Aquilegia coerulea PARG1a Aquca_125_00020.1

Arabidopsis lyrata PARG1a 482111

Arabidopsis lyrata PARG1b 902224

Arabidopsis lyrata PARG1c 869477

Arabidopsis thaliana PARG1 AT2G31870

Arabidopsis thaliana PARG2 AT2G31865

Betula pendula PARG1a evm.model.Contig2059.5

Betula pendula PARG1b evm.model.Contig3196.2

Brachybodium distachyon PARG1a Bradi1g01980

Brachybodium distachyon PARG1b Bradi1g01942

Brachybodium distachyon PARG1c Bradi3g35690

Brachybodium distachyon PARG1d Bradi3g48340

Capsella rubella PARG1a Carubv10022947m

Capsella rubella PARG1b Carubv10025449m

Capsella rubella PARG1c Carubv10025285m

Capsella rubella PARG1d Carubv10024816m

Cucumis sativus PARG1a Cucsa.266170

Eucalyptus grandis PARG1a Eucgr.A00416

Glycine max PARG1a Glyma11g01253

Glycine max PARG1b Glyma11g01260

Hordeum vulgare PARG1a F2DKT7_HORVD

Medicago truncatula PARG1a Medtr3g029520

Oryza sativa PARG1a LOC_Os03g62680

Physcomitrella patens PARG1a Phpat.003G047000

Physcomitrella patens PARG1b Phpat.004G067000

Populus trichocarpa PARG1a Potri.007G145800

Populus trichocarpa PARG1b Potri.017G002000

Prunus persica PARG1a ppa004452m

Selaginella moellendorffii PARG1a 77537

Selaginella moellendorffii PARG1b 84118

Solanum lycopersicum PARG1a Solyc12g017920

Solanum lycopersicum PARG1b Solyc12g096610

Solanum melongena PARG1a Manually annotated

Solanum melongena PARG1b Sme2.5_01701.1_g00005

Solanum tuberosum PARG1a Manually annotated

Solanum tuberosum PARG1b PGSC0003DMP400051135

Sorghum bicolor PARG1a Sobic.001G016000.

Spirodela polyrhiza PARG1a Manually annotated (Spipo7G0054700, Spipo7G0054800)

Theobroma cacao PARG1a Thecc1EG021532t1

Vitis vinifera PARG1a GSVIVT01038259001

Zea mays PARG1a GRMZM2G155935_T01

Homo sapiens PARG Q86W56

Documents

Plant PARPs, PARGs and PARP-like proteins