Characterization of wheat (Triticum aestivum) TIFY family and role of Triticum Durum TdTIFY11a in salt stress tolerance

The TIFY proteins constitute a plant-specific super-family and they are involved in regulating many plant processes, such as development, defences and stress responses. The Jasmonate-ZIM-Domain (JAZ) proteins, the best-characterized sub-group of the TIFY family are key regulator of the jasmonic acid (JA) signalling pathway. Jasmonates regulate several aspects of plant development, and play a primary role in defence mechanisms as well as in plant responses to abiotic stresses. The TIFY family is well studied in dicots but poorly investigated in monocots. The present study reports an extensive genomic identification of TIFY proteins from Triticum aestivum. We identified 49 TIFY genes, which were annotated according to three sub-genomes (AABBDD) of T. aestivum. Following their clustering with Oryza sativa and Brachypodium distachyon, the 49 genes were grouped in 18 different TIFY homeologous subsets. Expression analyses of 6 representative TIFY genes on Tunisian durum wheat seedlings revealed their differential regulation by various stress treatment, including JA, ABA and salt stress. TIFY11a was specifically induced after salt treatment. Transgenic lines over-expressing TdTIFY11a showed higher germination and growth rates under high salinity conditions, compared to wild type plants. In summary, our results outline a relevant role of wheat TIFY proteins in promoting germination under salt stress.


Introduction
Because of their sessile lifestyle, plants have evolved myriads of defense mechanisms to survive the continuous challenges of their ever-changing environment, including exposure to pathogens and insects but also, droughts, salty soils or mineral deficiency.Many signaling pathways participate in plant adaptation to environmental cues.Plant hormones are major actors of plant defense against environmental changes and among them abscisic acid (ABA) is considered as the abiotic stress hormone while jasmonic acid (JA) is traditionally regarded as the hormone that regulates plant defenses to necrotrophic pathogens, fungi, insect and nematodes [1][2][3].
All JAZ proteins retain the conserved ZIM (Zinc-finger expressed in Inflorescence Meristems) or TIFY domain, and therefore they belong to the plant specific family called TIFY family that includes JAZ, TIFY8, ZIM-like (ZML) and PEAPOD (PPD) proteins which have been particularly well studied in Arabidopsis [13].These Arabidopsis proteins all possess a conserved TIFY or ZIM domain composed of 36 amino acids containing a core motif TIF[F/Y]XG [13].The TIFY domain is required for JAZ dimerization and mediates the interaction with NINJA (Novel Interactor of JAZ), which recruits the TOPLESS (TPL) general transcriptional corepressor [11,14,15].
In addition to the TIFY domain, ZMLs possess a C2C2-GATA zinc-finger DNA-binding domain and a CCT-domain (CONSTANS, CO-like, TOC1) that is closely related to the Jas domain in JAZ proteins [16].In contrast, the PPD proteins, beside the TIFY domain, harbor at their N-terminus a typical PPD domain [13,17].Some TIFY proteins, such as AtJAZ7 and AtJAZ8, hold an EAR motif (ethylene-responsive element binding factor-associated amphiphilic repression) that enables them to directly recruit the TPL co-repressor [18].
Beyond Arabidopsis, TIFY families have been recently described in several plant species, including tomato, rice, maize and Brachypodium [13,[19][20][21][22]. Different functions have been described for TIFY proteins belonging to different subfamilies.For example, TIFY8, PPD and ZML proteins are involved in the transcriptional regulation of developmental processes.In Arabidopsis,loss-of-function mutations of PPD1 and PPD2 affect leaf shape, silique length modifications and meristemoid division [17,23], while the leguminous ortholog PPD gene BIG SEEDS1 regulates cell proliferation and plant organ size [24].AtTIFY1/ZML over-expression results in hypocotyl elongation while ZML2 acts as a transcriptional repressor in lignin biosynthesis in maize [16,25].In all plant species studied, JAZ proteins are the most represented groups in TIFY families.Arabidopsis possess 13 different JAZ members with extensive redundancy, but also specific functions [4].For instance, AtJAZ12 is specifically degraded after interaction with the ABA repressor-E3 Ubiquitin ligase KEG (KEEP on GOING) [26].AtJAZ2 is expressed only in stomata where it triggers stomatal closure to hinder pathogen penetration [27].
Wheat is one of the most consumed cereals worldwide and its production is highly sensitive to environmental constraints [34].Modulation of the JA pathway could be a novel strategy for biotechnological improvement of its productivity.However, little is known about the wheat TIFY proteins.Recently, 14 homeologous JAZ genes have been identified in Triticum aestivum L. [29] but a complete view of the wheat TIFY family is still lacking.Here, we provide a complete identification and characterization of Triticum aestivum TIFY protein family and the first evidence that the wheat JAZ/TIFY genes are involved in plant salt stress tolerance.

Plant material and stress treatments
Seeds of Tunisian durum wheat variety Oum Rabiaa3 provided from INRAT (Tunisian Agronomic Research Institute) were surface sterilized with 1.5% (v/v) sodium hypochlorite for 15 min with gentle agitation, rinsed three times with sterile water and grown on wet Whatman paper, for 2 days in the dark, and for a week in a growth chamber at 23˚C, under a 16 h photoperiod (16 h light/8 h dark) and 60% relative humidity.Stress treatments were done on ten 7-day-old seedlings using 150 mM NaCl, 50 μM JA, 100 μM ABA for 1 and 6 h.
Arabidopsis Col-0 seeds were obtained from the NASC Stock Center and used for transformation using the floral dip method [35].For salt tolerance tests, after seed surface-sterilization and vernalization for 2 days at 4˚C, seeds were grown on MS medium (0.5x, 0.7% agar) supplemented or not with NaCl (100, 150 or 200 mM).
Germination rates of 20 to 50 seeds were evaluated by observation of radicle emergence and cotelydon greening at 2 and 5 days after germination (DAG) respectively.Similar results were obtained in at least 4 independent biological replicates.
Root growth inhibition and accumulation of anthocyanins of 10-to-30 10-day-old seedlings grown in absence or presence of 50 μM JA were analyzed as described in [36].

Identification of Triticum aestivum TIFY gene family and phylogenetic analyses
Common wheat TIFY protein sequences were retrieved by combining HMMER, BLAST analyses using Oryza sativa and Brachypodium distachyon TIFY proteins [20,21] as query on TGACv1 genome from EnsemblPlant (http://plants.ensembl.org/Triticum_aestivum/Info/Index)and phytozome databases (https://phytozome.jgi.doe.gov) as well as keyword searches using TIFY and JAZ as queries.The retrieved proteins have been analyzed using Pfam (http://pfam.xfam.org/) to ensure the presence of the TIFY domain.
The wheat TIFY proteins were aligned using MEGA 6.06 together with Brachypodium distachyon and Oryza sativa TIFY proteins [37].Based on multiple alignment (CLUSTALW, Blosum matrix with default settings), pairwise comparison and phylogenetic analyses, we assigned to the 49 wheat different proteins their TIFY name.The phylogenetic tree was constructed using MEGA6.06 and the Neighbor-end joining method based on the number of aa substitutions.

RNA extraction and gene expression analyses
Wheat total RNA extraction was performed on aerial parts of ten 7 day-old seedlings of durum wheat variety Oum Rabiaa3 treated as above-mentioned using Trizol reagent (Invitrogen) with manufacturer's recommendations.The RNA was cleaned up from DNA contamination using on-column DNAse I removal kit (Roche). 1 μg of total RNA was used for reverse transcription using cDNA synthesis kit (Roche).After 1/10 th dilution, 5 ml of cDNA was used as a template for QPCR analyses in a total volume of 15 ml using Power SYBR Master mix (Applied Biosystems) as previously described [38].Amplification and quantification was performed in a 7500 Real Time PCR system (Applied Biosystems).Wheat Actin gene (TRIAE_CS42_1AS_T-GACv1_020044_AA0074210) was used as internal control.Quantification was performed using the ΔΔCt method [39] using actin and time 0 as references.Actin and TIFY primer pairs are reported in S1 Table .A RNA isolation kit (FavorGen) was employed to extract Arabidopsis total RNA using biological samples of tissue pooled from 10-15 5-day-old seedlings.RNA was extracted including DNase digestion to remove genomic DNA contamination.cDNA was synthesized from 1.5 μg total RNA with the high-capacity cDNA reverse transcription kit (Applied Biosystems).For gene amplification, 4 μl from a 1:10 cDNA dilution was added to 4 μL of EvaGreen1 qPCR Mix Plus (Solis BioDyne) and gene-specific primers previously described [38].Quantitative PCR was performed in 384-well optical plates in a HT 7900 Real Time PCR system (Applied Biosystems) using standard thermo cycler conditions (an initial hold at 95˚C for 10 min, followed by a two-step SYBRPCR program of 95˚C for 15 s and 60˚C for 60 s for 40 cycles).Relative expression values are the mean ± SD of three to four technical replicates relative to the basal wild-type control using ACT8 as housekeeping gene.

TdTIFY11a isolation and cloning
Using cDNA sequences of Triticum aestivum TaTIFY11a, primers were designed for PCR amplification of either the complete ORF or a truncated form lacking the Jas domain (ΔJas).For the full-length TIFY11a cloning, a first PCR amplification using JAZ2bisF1 (5'-CGGTTGG TGGAGTGCTTAGC-3') and JAZ2bisR1 (5'-TGTACCAACGTTGCCGTGCA-3') was done on wheat cDNA of Oum Rabiaa3 Tunisian durum wheat variety by adding 1% DMSO using the following program: 94˚C, 30 s; 58˚C, 30 s; 72˚C 1 min.One microliter of this first 625bp-PCR product was used for nested PCR amplification using JAZ2bisF2 (5'-AAGGCCATCGATCGCC ACCG-3') and JAZ2bisR2 (5'-TGTTGAGGCGATCATTCACG-3') and an annealing temperature of 58˚C.A single 584 bp-band was observed and cloned into the pGEMTeasy vector (Promega) giving rise to the pTIFY11a-FL clone which was then confirmed by sequencing using the dye terminator cycle sequencing method (Applied Biosystems).

Protein extraction and western blotting
A minimum of 20 mg of seedlings were collected and frozen in liquid nitrogen before quick grinding in sample buffer (0.5 M Tris-HCl (pH 8.5), 4% (w/v) lithium dodecyl sulfate, 20% (v/ w) glycerol, 1 mM EDTA, 0.25 M DTT and tracking dye) to extract total proteins.The extraction was followed by 15 min centrifugation at 13 000 rpm and boiling at 100˚C.The proteins were then separated on a 12% SDS-PAGE.After transfer on nitrocellulose membrane using the Mini-transfer system (BioRad) for one hour at 100 V, the blot was blocked during one hour in PBS, 5% milk.Then, the blot was incubated with anti-GFP antibody HRP conjugated (1/1000) for 1 hour.Detection was performed using the West Femto chemiluminescent signal detection kit (Pierce).Equal loading of total proteins was assessed by blotting the same membrane with mouse anti-actin antibody (1/2000) for 1 hour in PBS, 0.05% milk followed by incubation with anti-mouse IgG-HRP conjugated (1/10000; Roche).Detection was performed using the micro chemiluminescent signal detection kit (Pierce).

Bioinformatic tools and statistical analyses
MEME suite (http://meme-suite.org/tools/meme)was used with default settings to identify conserved motifs within TIFY proteins.TIFY proteins were represented on scale using GPS1.0 drawing tool.
Statistical analyses were performed using One-way ANOVA with post-hoc Tukey HSD Test for comparing multiple treatments.

Common wheat TIFY protein family
The different members of common wheat TIFY family proteins were retrieved by performing BLAST searches on Uniprot (http://www.uniprot.org)and Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html)databases using available protein sequences of rice and Brachypodium TIFY proteins [20,21].These searches allowed us to identify 49 T. aestivum TIFY genes, of which 15 were novel.Following their clustering with rice and Brachypodium, these 49 TaTIFY genes were grouped into 16 homeologous loci, with one gene copy on each of the three wheat subgenomes (T.aestivum AABBDD), and annotated accordingly (ie.-A; -B; -D) (Fig 1 and Table 1).
Phylogenetic analyses identified 4 groups within the 18 TaTIFY proteins (Fig 1 and Table 1).The phylogenetic tree revealed that 4 major clades of TIFY proteins are present in the 3 monocots (wheat, rice and Brachypodium) (Figs 1 and 2A).Proteins in the TIFY3, TIFY5/6 and TIFY10/11 groups possess, in addition to the typical TIFY motif (Figs 2B and S1), the canonical Jas domain characteristic of the JAZ repressors (Figs 2C and S2).Proteins in group TIFY1/2 (TaTIFY1a, 1b, 2a, 2b in the case of wheat) possess, besides the TIFY motif, a CCT domain and a C2C2-GATA-Zinc finger DNA binding domain, which are typical of ZIM-subfamily proteins (Figs 2D and S3).The PEAPOD domain is typical of the TIFY4 family in Arabidopsis [13,23] but no proteins showing similarity to AtTIFY4 have been found in any of the studied monocot species (wheat, rice or Brachypodium).Finally, we did not find in T. aestivum any ortholog of TIFY8 as observed for Brachypodium [20,21].
The analysis of the genomic localization of wheat TIFY genes showed that the 18 groups are located on chromosome 2 (n = 4), 4 (n = 5), 5 (n = 3), 6 (n = 2) and 7 (n = 4) and distributed along the three subgenomes.Only one gene, TaTIFY11f-U, could not be assigned to any specific chromosome (U).Recently, Wang et al., [29] described 34 different common wheat TaJAZ genes grouped in 14 homeologous subsets, which possess Jas and TIFY domains.Within the 18 TIFY homeologous proteins identified here, the 14 homeologous JAZ have been retrieved and classified in three distinct groups (JAZ1, JAZ2 and JAZ3).However, the previously named TaJAZ4, TaJAZ5, TaJAZ11 and TaJAZ14 exhibit a TIFY domain but a divergent CCT domain, not a canonical Jas domain [29].In addition, these TaJAZ proteins retain a GATA domain and should therefore be classified as ZIM-like proteins rather than JAZ proteins (Fig 2) [13].Within the TIFY3, TIFY5/6 and TIFY10/11 groups of wheat JAZ proteins, the Jas domain is highly conserved (

Expression analyses of wheat TIFY genes under stress conditions
Transcriptional regulation of JAZ genes in response to abiotic stresses has been reported in several plant species [19][20][21]28,29].To assess the expression of durum wheat JAZ/TIFY genes under various stress treatments, we selected six wheat genes orthologs of monocot salt-induced JAZ/ TIFY genes [20,21].Expression analyses were performed by qRT-PCR on the well-characterized Oum Rabiaa Tunisian durum wheat variety after either 1 or 6 hours exposure to JA (50 μM), ABA (100 μM), or NaCl (150 mM).As shown in Fig 3 , TdTIFY10c, TdTIFY11a, TdTIFY11c and TdTIFY11f were quickly induced by salt treatment.This induction is transient since 6 hours after salt treatment the basal level of TdTIFY expression is restored, with the exception of TdTI-FY10c, which is still slightly induced (Fig 3).Among the tested genes, TdTIFY11a showed the strongest expression in response to salt.In contrast, salt stress did not alter TdTIFY3 expression, whereas it slightly down-regulated TdTIFY6.JA treatment up-regulated all tested JAZ/TIFY genes except TdTIFY6b (Fig 3).ABA down-regulated the expression of most of the genes with the exception of TdTIFY3 and TdTIFY6b (Fig 3).In summary, this analysis reveals that TdTIFY genes are differentially regulated in response to salinity and hormone treatments.

Identification and characterization of TdTIFY11a
TdTIFY11a is highly induced by salt, mildly up-regulated in response to JA and not induced by ABA.The TdTIFY11a ortholog OsTIFY11a/OsJAZ9 exhibited a similar expression pattern and its overexpression conferred salt and drought stress tolerance in rice transgenic plants [20].Therefore, we analyzed the putative role of TdTIFY11a in salt-stress responses.The TdTIFY11a gene from Triticum durum Oum Rabiaa variety was isolated.Its nucleotide sequence was 99, 93 and 91% identical to common wheat genes TaTIFY11a-B, TaTIFY11a-A and TaTIFY11a-D, respectively.The TdTIFY11a encoded protein is 100% identical to TaTIFY11a-B but only 80% to TaTIFY11a-D and TaTIFY11a-A (S5 Fig) .Next, two GFP-tagged TdTIFY11a constructs (the full-length sequence or a truncated version without the Jas domain) were expressed in Arabidopsis plants under the constitutive CaMV 35S promoter.Two lines for each construct were chosen based on the highest TdTI-FY11a-GFP protein accumulation (ie.lines 8 and 17 for the full-length version and lines 40 and 57 for the ΔJas construct) (S6 Fig) .The phenotypes of these transgenic lines were compared to wild type (WT) under control and salinity conditions.Seeds were germinated in the presence of 100 and 150 mM NaCl concentrations.Germination rates were measured as radicle emergence 2 days after germination, whereas cotyledon greening was recorded 5 days after germination.Under control conditions, the full-length TdTIFY11a-GFP and TdTIFY11aΔJas-GFP lines germinated equivalently to WT control (Fig 4A -4B).However, in presence of salt all the TIFY11a over-expressing lines exhibited significantly higher germination rates compared to WT seeds (Fig 4A -4B).This enhanced salt tolerance is more pronounced on full-length TdTIFY11a-GFP than TdTI-FY11aΔJas-GFP seedlings.For example, in the presence of 150 mM NaCl, both TdTIFY11a-GFP lines had 3-5 fold increases, while TdTIFY11aΔJas-GFP lines showed only 2 fold increase in radicle emergence compared to WT.The difference in radicle emerge of TdTIFY11a line 17 is significantly higher than that of wild-type seedlings (p-value <0.01; Fig 4B ).A significant (30%) increase in cotyledon greening was also observed at day 5 on the transgenic lines germinated on salt containing medium in comparison with wild-type control plants (Fig 4C).The TdTIFY11aΔJas-GFP line 17 showed the highest cotyledon greening ratio (50%) at 150 mM (Fig 4C).To confirm that TdTIFY11a-GFP and TdTIFY11aΔJas-GFP proteins still accumulate after few days of germination in presence of salt, the levels of the TdTIFY11a-GFP were monitored; the truncated TdTIFY11aΔJas-GFP is detected at similar levels in the absence or presence of salt stress treatment.In the case of full length TdTIFY11a-GFP, less protein seems to accumulate after germination in high salinity conditions (S6 Fig) .Next, we reasoned that TdTIFY11a overexpression could influence the expression of endogenous AtJAZ levels.For this purpose, the expression in the four AtJAZ genes in TdTIFY11a transgenic lines was analyzed by qRT-PCR in control or salt stress conditions (see S7 Fig) .In basal conditions, levels of AtJAZs in OE-TdTIFY11a transgenic lines are Altogether, the results show that the over-expression of full-length and truncated TdTI-FY11a confers higher germination rates under high salinity conditions.

Discussion
Plant adaptation to their changing environment is orchestrated by complex regulatory networks where JA-Ile plays a primary role in regulating defense mechanisms and abiotic stress responses [2,4,28].JA-Ile acts through a well-described signaling pathway, in which JAZ proteins are central negative regulators of JA responses [7,9].The JAZ family belongs to the larger TIFY super-family, well characterized in eudicots such as Arabidopsis thaliana but still poorly known in wheat.To date, 14 homeologous JAZ loci have been identified in the common wheat (Triticum aestivum L.) and their expression patterns characterized in response to stress treatments [29].However, the identification of the complete TIFY super-family in wheat was lacking.Our study identifies 49 TIFY proteins encoded by 49 genes located in the three different wheat subgenomes.The identification in wheat of all orthologous proteins of rice and Brachypodium indicates exhaustiveness of our analysis.Phylogenetic and domain analyses show that TaJAZ4, TaJAZ5, TaJAZ11 and TaJAZ14 [29] contain a CCT motif and GATA motif typical of ZIM-like proteins; therefore TaJAZ4, TaJAZ5, TaJAZ11 and TaJAZ14 are not "bona-fide" JAZ proteins and should be best referred as TaTIFY1a, TaTIFY1b, TaTIFY2a and TaTIFY2b respectively (Fig 1).
No orthologous protein of AtTIFY8 could be identified in wheat and Brachypodium.Likewise no TIFY7 could be identified in Brachypodium, rice and wheat, indicating that these classes of proteins might be specific of eudicots but absent in monocots.Five wheat TIFY proteins contain a canonical EAR motif (LxLxL) (Figs 2 and S4), supporting the hypothesis of a direct recruitment of TPL to negatively regulate JA-mediated transcription and the conservation in wheat of the JAZ-TPL repression mechanism [40].Wheat orthologous of PPD proteins were not identified, in agreement with their absence in rice and Brachypodium, supporting the hypothesis that the PPD subfamily is only present in dicots [41].
The Jas motif of the wheat JAZ is highly conserved (Figs 2C and S2), including the specific residues directly mediating COI1-JAZ complex formation, hormone binding and JAZ interaction with MYC TFs [6,12].This high conservation of the functional residues within the Jas motif suggests that wheat JAZ proteins are able to interact with the corresponding key wheat JA-pathway components in a similar fashion as described in Arabidopsis.
The expression pattern of six TdTIFY genes showed that they are differentially regulated by JA, ABA and salt (Fig 3).Interestingly, their regulation is comparable to that of the rice and Brachypodium orthologous TIFY genes.For instance, the three monocot TIFY11a orthologous genes are all up-regulated by salt, slightly induced by JA but not affected by ABA [20,21] (this work).Hence, their expression might be mediated by conserved regulatory mechanisms.
The expression of OsTIFY11a/OsJAZ9 under drought-inducible promoter confers drought and salt stress tolerance to rice plants, without altering the responses to JA [20,21].Likewise, over-expressing two durum wheat ortholog TdTIFY11a variants in Arabidopsis does not alter responses to JA but increases germination efficiency under salt stress conditions, including higher radicle emergence rates and enhanced seedling establishment (Figs 4C, S8 and S9).These are important agronomic traits in the context of abiotic stress tolerance-ie.seeds are able to germinate despite adverse conditions.Both transgenic lines over-expressing either full-length TdTI-FY11a or the truncated TdTIFY11aΔJas, are similarly salt stress tolerant, suggesting that the Jas domain may not be critical for the positive regulatory role of TIFY11a in salt stress tolerance.However, TdTIFY11aΔJas does retain the ZIM/TIFY domain mediating the interaction of several AtJAZ proteins with AtWRKY57, whose over-expression confers salt tolerance in Arabidopsis plants [42,43].Similar to the case of TdTIFY11a over-expression plants, the AtWRKY57-mediated stress tolerance only occurs in seed germination and early post-germination growth, whereas adult plants fail to show salt tolerance.This suggests that the role of TdTIFY11a in salt tolerance may rely on the activity of the wheat WRKY57 orthologs.Besides, salt and drought tolerance conferred by OsTIFY11a/OsJAZ9 over-expression was reported only in young rice seedlings [20], similarly to the case of TdTIFY11a over-expression plants.Several OsJAZ proteins directly interact with OsbHLH148, which in turn modulates the expression of JA-regulated ion transporters and promotes stress tolerance [31,44].In addition, OsTIFY11a/OsJAZ9 also interacts with and regulates OsbHLH062, a TF that directly binds to the promoters of the ion transporter genes such as OsHAK21 to regulate salt tolerance in rice plants [31].It is therefore reasonable that TdTIFY11a may act in a similar manner in Arabidopsis, conferring salt stress tolerance via the OsbHLH148 and/or OsbHLH062 orthologous-signaling pathway.However, the truncated TdTIFY11aΔJas lacking the Jas motif would not directly interact with these bHLH TFs.It is feasible that the TdTIFY11aΔJas variant would dimerize with additional JAZ proteins and consequently indirectly interfere with these or other TFs.Future identification and characterization of the orthologous wheat bHLH148 and/or OsbHLH062 orthologous will test this hypothesis.
On another hand, heterologous expression of TdTIFY11a constructs may interfere with the endogenous expression of AtJAZ genes, which in turn could confer germination tolerance in high salinity conditions.In basal conditions, the endogenous levels of JAZs in TdTIFY11a and TdTIFY11aΔJas transgenic lines are very similar to those in wild-type seedlings, providing evidence against the hypothesis that altered basal JAZ expression may prime germination tolerance (S7 Fig) .As previously reported, most JAZ genes are induced in response to high salinity stress.This salt-induction of JAZ genes is generally higher in TdTIFY11a transgenic lines; therefore, it is plausible that this enhanced JAZ expression may depend on the ectopic TdTI-FY11a over-expression.However, the enhanced JAZ expression in the TdTIFY11a transgenic lines is not very high, approximately twice that of wild-type plants (S7 Fig) .Therefore, the hypothesis that variation in JAZ expression may affect the salt tolerance response in OE-TdTI-FY11a transgenic lines requires further studies.
The rice RSS3 protein forms a ternary complex with OsbHLH094 and OsTIFY11a/OsJAZ9 [45].OsRSS3 and OsTIFY11a synergistically regulate the expression of JA-induced salt-responsive genes [45]; therefore the enhanced salt tolerance of TdTIFY11a over-expressing plants may also involve the orthologous RSS3 wheat gene.Finally, OsTIFY11a/OsJAZ9 also interact with additional TFs involved in tolerance to stresses other than drought; for example, OsTIFY11a directly interacts with and represses OsMYB30, a key TF regulating cold tolerance in rice [46].Thus, it is reasonable that TdTIFY11a may regulate additional, still unidentified wheat TFs to mediate salt stress tolerance.
Why the over-expression of TdTIFY11a exhibits enhanced salt tolerance only at early stages of plant development (ie.seedling establishment) is unclear.The quicker turnover of TdTIFY proteins in mature tissues compare to early stage seedlings may account for the lack of stress tolerance in adult plants.Alternatively, specific spatiotemporal expression (ie.only expressed at seedling stage) of different TFs regulated by TdTIFY11a may explain the developmental specificity.
Plants growth under high salt stress conditions show partial decreases of full-length TdTIFY11a but not of TdTIFY11aΔJas protein level (S6 Fig) .In this context, salt stress induces accumulation of JA-Ile in plants [47][48][49].Therefore, the differential protein stability between TdTIFY11a and TdTI-FY11aΔJas may depend on the salt-induced accumulation of JA-Ile that in turn triggers full-length TdTIFY11a degradation.In contrast, the stability of TdTIFY11aΔJas (lacking the Jas motif mediating JA-Ile dependent COI1 interaction) is not affected by salt stress.
In conclusion, we identified 49 typical TIFY genes, grouped into 16 homeologous loci, in common wheat divided into two subfamilies, namely ZML and JAZ.Over-expression of TdTI-FY11a in Arabidopsis conferred higher germination rates under high salinity conditions indicating a relevant role of JAZ proteins in abiotic stress responses.Wild-type (Col-0) and transgenic TdTIFY11a seedlings (N = 10 to 30) were germinated in absence (control) or presence of 50 μM JA.Nine days after germination, root growth (A) (mm) and anthocyanin accumulation (B) [Abs(530nm)/fresh weight (mg)] were measured.coi1-1 seedlings were included as control.Data presented as box-plots; horizontal lines are medians, boxes show the interquartile range and error bars show the full data range.The experiments were repeated at least 2 times with similar results.Letters stand for statistical differences (One-way ANOVA with post-hoc Tukey HSD, p<0.01).(PDF)

Fig 1 .Table 1 .
Fig 1. Phylogenetic tree of common wheat, Brachypodium and rice TIFY proteins.Phylogenetic tree was obtained using MEGA6.06 with the Neighbor-Joining method based on TIFY protein sequences.Wheat, Brachypodium and rice gene identifiers are indicated such as in Table 1.Wheat proteins are indicated by red dots, rice proteins by dark blue squares and Brachypodium proteins by green triangles.The red open circle represent durum wheat TdTIFY11a.Scale bar indicates evolutionary distances inferred using the Neighbor-Joining method calculated by the number of amino acid substitutions per site as conducted by MEGA6.06.https://doi.org/10.1371/journal.pone.0200566.g001

Fig 2 .Fig 3 .
Fig 2. Conserved domains in wheat TIFY proteins.Schematic representation of 49 wheat TIFY proteins and conserved domains drawn with GPS tool (A).Blue boxes represent the TIFY domain, yellow highlight the Jas domain, green stand for divergent CCT motifs and red represent C2C2-GATA-Zinc-finger DNA binding domain.EAR motif is shown by black box.Grey bars represent non-conserved sections.The scale at the bottom (indicating the number of amino acid) corresponds to the proteins length.Consensus of sequences conservation of TIFY/ZIM domain (B), Jas motifs (C) and GATA domain (D) using MEME.https://doi.org/10.1371/journal.pone.0200566.g002

Fig 4 .
Fig 4. Over-expression of TdTIFY11a variants confers salt tolerance to Arabidopsis seedlings.(A) 7-day-old seedlings (N = 20 to 50) of the different over-expressing lines germinated on control media or in presence of 100 or 150 mM NaCl.Percentage of radicle emergence of 2 day-old seedlings (B), percentage of seedlings with green cotyledons at day 5 after germination (C) and on control media or supplemented with NaCl (100 or 150 mM).Data presented as box-plots; horizontal lines are medians, boxes show the interquartile range and error bars show the full data range.The experiments were repeated at least 4 times with similar results.Asterisks (B-C) indicate statistical significance (One-way ANOVA with post-hoc Tukey HSD, Ã p<0.05,ÃÃ p<0.01).https://doi.org/10.1371/journal.pone.0200566.g004 used for the QRT-PCR.(PDF) S1 Fig. Multiple sequence alignment of the conserved TIFY domain of several wheat TIFY proteins.Alignment of the 49 Triticum aestivum TIFY proteins showing the conserved TIFY domain.Protein IDs indicated are the same as listed in Table 1.The alignment was performed with MEGA6.06 using CLUSTALW and the BLOSUM matrix.(PDF) S2 Fig.Multiple sequence alignment of the canonical Jas motif of several wheat TIFY proteins.The alignment of the sequences of the conserved Jas motif of canonical wheat JAZ proteins (of the TIFY3, TIFY5/6 and TIFY10/11 clades) were employed (A).The alignment was performed with MEGA6.06 using the BLOSUM matrix.(B) Sequence logo of the Jas motif using MEME on the same proteins aligned in A. (PDF) S3 Fig. Multiple sequence alignment of the CCT motif and GATA domain of several wheat TIFY proteins.The alignment of the sequences of the conserved CCT motif (A) and GATA domain (C) of wheat TIFY proteins belonging to the group TIFY1/2 were employed.The sequence logo for the CCT motif (B) and GATA domain (D) were generated with MEME.(PDF) S4 Fig. Multiple sequence alignment of the EAR motif in wheat TIFY proteins.The alignment of the sequences of the conserved EAR motif (A) of five wheat TIFY proteins belonging to the group TIFY5 and TIFY11 were employed.(B) The sequence logo for the EAR motif.(PDF) S5 Fig. Alignment of TaTIFY11a and TdTIFY11a sequences.A) Multiple protein alignment of TaTIFY11a-A, -B, -D and TdTIFY11aperformed with MEGA6 (MUSCLE matrix).Residues highlighted in blue are conserved among all proteins whereas residues in red are conserved between TaTIFY11a-B and TdTIFY11a.B) Phylogenetic tree performed with MEGA6 based on the multiple alignment in A using Neighbour-joining method with BLOSUM matrix and 1000 bootstrap iterations.C) Multiple cDNA alignment performed with MEGA6 (MUSCLE matrix).Nucleotides highlighted in blue are conserved among all genes.Nucleotides marked in red are conserved between TaTIFY11a-B and TdTIFY11a, whereas the only two divergent nucleotides between TaTIFY11a-B and TdTIFY11a are highlighted in yellow.(PDF) S6 Fig.Protein accumulation of TdTIFY11a-GFP variants.Immunoblot analyses of TdTI-FY11a-GFP and actin protein levels in 35S:TdTIFY11a-GFP (full length, line 8 and 17), TdTI-FY11aΔJas-GFP (line 57); wild-type Col-0 (WT) was included as a negative control.Seeds were germinated in control media (-) or in presence of 100 mM NaCl (+) and seven-day-old seedlings were used for the analysis.Protein molecular weights are indicated at the sides.(PDF) S7 Fig. JAZ gene expression in TdTIFY11a transgenic lines.Gene expression analysis of JAZ genes in 5-day-old Arabidopsis seedlings treated with mock solution or 150 mM NaCl.Relative expression of JAZ genes was analyzed by quantitative real-time qPCR using actin 8 as housekeeping control.Each biological sample consisted of tissue pooled from 10-15 plants.Data show mean ± SD of three to four technical replicates.(PDF) S8 Fig. Abiotic stress responses of adult TdTIFY11aΔJas-GFP plants.3-week-old plants wild-type and TdTIFY11aΔJas-GFP (line 57) were exposed to increasing salt concentrations (100 to 400 mM NaCl) or drought stress.Wild-type and TdTIFY11aΔJas-GFP showed similar responses to these abiotic stresses.(PDF) S9 Fig. Analyses of over-expression of TdTIFY11a variants in response to JA treatment.