An updated census of the maize TIFY family

The TIFY gene family is a plant-specific gene family encoding a group of proteins characterized by its namesake, the conservative TIFY domain and members can be organized into four subfamilies: ZML, TIFY, PPD and JAZ (Jasmonate ZIM-domain protein) by presence of additional conserved domains. The TIFY gene family is intensively explored in several model and agriculturally important crop species and here, yet the composition of the TIFY family of maize has remained unresolved. This study increases the number of maize TIFY family members known by 40%, bringing the total to 47 including 38 JAZ, 5 TIFY, and 4 ZML genes. The majority of the newly identified genes were belonging to the JAZ subfamily, six of which had aberrant TIFY domains, suggesting loss JAZ-JAZ or JAZ-NINJA interactions. Six JAZ genes were found to have truncated Jas domain or an altered degron motif, suggesting resistance to classical JAZ degradation. In addition, seven membranes were found to have an LxLxL-type EAR motif which allows them to recruit TPL/TPP co-repressors directly without association to NINJA. Expression analysis revealed that ZmJAZ14 was specifically expressed in the seeds and ZmJAZ19 and 22 in the anthers, while the majority of other ZmJAZs were generally highly expressed across diverse tissue types. Additionally, ZmJAZ genes were highly responsive to wounding and JA treatment. This study provides a comprehensive update of the maize TIFY/JAZ gene family paving the way for functional, physiological, and ecological analysis.


Introduction
Jasmonates (JAs) are plant oxylipin hormones involved in the regulation of diverse physiological processes in plants, including reproductive development, abiotic stress response, and defense against insect and microbes [1][2][3]. In plant cells, jasmonates are synthesized from linolenic acid via the octadecanoid pathway [4][5][6], through the activity of at least eight enzymes (lipase, lipoxygenase, allene oxide synthase and cyclase, 12-OPDA (12-oxophytodienoic acid) reductase, acyl-CoA oxidase, a multifunctional protein, and 3-ketoacyl-CoA thiolase) [7][8][9]. JA perception occurs through the interaction of the biologically active ligand, JA-Ile, with of total TIFY genes where as little as 27 to as high as 48 were reported [15,[40][41][42]. In 2016, the maize reference genome was updated using single-molecule sequencing technology to Zm-B73-REFERENCE-GRAMENE-4.0 (also known as "B73 RefGen_v4" or "AGPv4") which is substantially different from the previous AGPv3. In this study, we utilized version 4 of the maize reference genome to update the list of TIFY genes and classified them into subfamilies based on the presence of their respective conservative domains. To provide insights into the functions of different family members, the expression of all the ZmTIFY genes were assessed in various tissues and organs at different developmental stages and in response to wounding and JA chemical treatment. In addition, the promoters of ZmTIFY genes were analyzed for predicted cis-elements that may explain potential conditional-dependent gene induction.

Plant material
The maize inbred B73 was used as the plant material for this study. The seeds were sowed in plastic boxes containing a soil mix of vermiculite: organic substrate: loam (1:1:1 v/v/v). The seedlings were grown in a greenhouse at 25-35 C with relative humidity maintained at 60%-85% and illuminated by natural sunlight. The experiments were carried out in the seasons of Spring or Autumn when the average photoperiods were approximate 12 h-day/12 h-night.

Mechanical wounding and JA treatment
The mechanical wounding treatment was conducted as described by [44]. The second leaf of a V3 stage plants was squeezed with pliers twice on each side of the midrib about 1cm apart without damaging to the midrib. The undamaged midsection flanked by the two wound sites was collected at 0, 1, 3, 6 h post-wounding and frozen immediately in liquid nitrogen and stored at -80˚C for downstream analysis. Seedlings at the V3 stage were sprayed with 100μM of JA solution or water as control until both sides of the leaves were completely wet and collected at 0, 6, 12, 24, and 48 h after chemical treatment, frozen immediately in liquid nitrogen, and stored at -80˚C until further analysis. Three biological replicates were collected per time-point for each treatment-group.

Gene expression analysis
Total RNA was extracted using Trizol according to the manufacturer's instructions and its integrity was tested on a 1% agarose gel by visualizing defined 16S and 18S rRNA bands. Genomic DNA was removed according to Goldenstar TM RT6 cDNA synthesis kit (Sangon Biotech Co. Ltd at Shanghai). For reverse transcription, 2 μg of total RNA was used to generate cDNA through the Goldenstar TM RT6 cDNA synthesis kit according to the manufacturer's instructions. The cDNA synthesis reactions consisted of 2μl (~2μg) of RNA template, 4μl of Goldenstar TM RT6 cDNA synthesis mix, and 14μl of RNase-free water followed with incubation at 50˚C for 30 minutes and then at 85˚C for 10 minutes.
Expression analysis was conducted with semi-quantitative real-time PCR using primers designed to selectively amplify distinct JAZs (S3 Table) and EIF4A gene was used as the housekeeping gene control for equal loading of cDNA. The reaction consisted of 12.5μl of 2xTaq PCR master mix (Sangon Biotech Co. Ltd at Shanghai), 1 μl forward primer (10μM), 1μl reverse primer (10μM), 1 μl (100 ng) of cDNA and ddH2O to a final volume of 25μl. Thermal cycling conditions were: 94˚C for 4 mins; 94˚C for 30 s, 57-58˚C for 30 s, and 72˚C for 30 s, a final incubation at 72˚C for 10 min, and depending on reaction, 28-30 cycles were performed. The PCR products were separated and visualized by gel electrophoresis on a 2% agarose gel.

Identification of the maize TIFY gene family and domain analysis
To identify the members of the TIFY family in maize, BLASTP searches were performed on the maize genome database (B73 RefGen_v4, https://maizegdb.org/) using the amino acid (AA) sequences of TIFY and Jas domains from TIFY proteins from Arabidopsis and rice as the search queries. Maize TIFY candidate genes were selected based on the criteria of 50% or greater AA identity and an e-value of 1e-4 or less. To determine the presence of the canonical TIFY subfamily domains, the predicted AA sequences of the ZmTIFY genes were submitted to the Pfam database (http://pfam.xfam.org/). For the analysis of the presence of an EAR (ERFassociated amphiphilic repression) motif, candidate proteins were manually compared to the previously reported 158 LxLxL-types of EAR motifs [45].

Tissue-specific expression profiling
RNA-Seq data for tissue-specific expression in 79 tissues [46] were obtained from maizegdb. org. The expression heatmap for tissue-specific expression was created by the software HemI 1.0 [47] using log 2 value of FPKM (fragment per kilobase per million mapped reads) of ZmTIFY genes.

Phylogenetic analysis of TIFY genes
A multiple protein sequence alignment was performed for the TIFY family members of Arabidopsis, maize, and sorghum using the online software MUSCLE (www.ebi.ac.uk/Tools/msa/ muscle/). The phylogenetic tree for all identified TIFY family genes in this study and for all known JAZ genes in Arabidopsis and sorghum were generated with the MEGA 7.0 software using the maximum likelihood method and robustness tested by bootstrapping for 1000 times. The tree was displayed using the online software Evolview v3 [48].

cis-element identification in promoters of TIFY genes
To analyze the putative cis-acting elements of the promoters of the ZmJAZ genes, 1.5 kb of nucleotide sequence upstream of the start codon for each ZmJAZ gene was scanned in the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

The maize genome houses 47 bona fide TIFY family members including 16 newly identified members
To identify all TIFY genes in the maize genome, the B73 RefGen_v4 genome was surveyed by BLASTP for similar sequences to the AA sequences of the TIFY and Jas domains from the Arabidopsis and rice TIFY proteins. This analysis revealed 47 distinct gene models whose predicted proteins contain a TIFY or Jas domain (Tables 1 and S1). Among these, four were predicted to belong to the ZML subfamily and contained a TIFY, CCT, and GATA zinc finger domain, but no Jas domain. Five of 47 were predicted to belong to the TIFY subfamily which contains solely the TIFY domain (Tables 1 and S1). No PPD proteins were identified. The remaining 38 TIFY proteins were characterized as JAZ proteins, six of which had no TIFY domain at the N-terminus, but all had a Jas domain at the C-terminus (Tables 1 and S1). In total, 38 JAZ, 4 ZML, 5 TIFY-subfamily, and no PPD genes were identified in the maize genome B73 RefGen_v4. Among the 47 TIFY genes, nearly 40% have never been identified in previous analyses of the maize TIFY family. These 16 genes include 13 ZmJAZs, two ZmTIFYs and one ZmZMLs (Tables 1 and S1).

JAZ proteins are asymmetrically distributed between the two maize subgenomes
The 47 TIFY genes were found differentially distributed across the ten maize chromosomes. The four ZML genes were located on the chromosome 1, 5, and 6 and the five TIFY-subfamily genes were found on chromosome 1, 2, 4, and 9. The remaining 38 JAZ genes were found on distributed across all ten chromosomes (Fig 1; Table 1). Chromosome 1 was found to contain nine JAZ genes, six of which were clustered in two loci: ZmJAZ1, 2, and 27 and ZmJAZ4, 5, and 6. ZmJAZ14, 15, and 26 were clustered at the short arm of Chromosome 5. Maize is a paleopolyploid plant, which harbors two subgenomes (maize1 and maize2) where each constitutes a genome orthologous to the entire sorghum genome [49]. Interestingly, 32 TIFY genes were found in the maize1, 14 in the maize2, and only one in the region between the two subgenomes (Fig 1) compared to the 19 SbTIFY genes thus so far predicted in the sorghum genome [15].

Conserved protein domains and their features different across the maize TIFY family
All ZmTIFY genes are predicted to encode at least one protein which range in sizes from 60 AA to 1604 AA, however the vast majority of TIFY proteins are smaller than 300 AA (S1 Table). The candidate maize TIFY proteins were analyzed for their TIFY and Jas domain compositions along with screening for presence of EAR-motifs. The TIFY domain, also known as the ZIM domain, mediates homo-and heteromeric interactions between JAZ proteins [17,50] and it is necessary for binding to the NINJA-TPL repressor complex [18]. The C-terminal Jas domain is essential for the interaction of JAZ proteins [12] with the LRR domain of JA receptor, COI1 protein [51]. The EAR (ERF-associated amphiphilic repression) motif is a principle mechanism of plant gene regulation and facilitates recruitment of TPL for transcriptional repression [24]. The analysis revealed that all but seven TIFY proteins contained both the TIFY and the Jas domains. The TIFY motif was absent in ZmJAZ 29, 30, 31, 32, and 36, and while it was truncated in ZmJAZ25 and ZmZML4 (S1 Table; Fig 3). JAZ4, 10, and 14 had incomplete Jas domains (S1 Table) and lacked the X5PY. X5PY motif required for JAZ degradation via 26S proteasome [12]. The Jas domains of ZmJAZ13, 17, and 26 had VPQAR in place of the normal LPIAR degron motif the sequence signal required for JAZ repressor degredation [24]. Manual sequence analysis uncovered that seven ZmJAZs (ZmJAZ4, 5,6,15,23,26 and 34) along with ZmTIFY3 and 5 possessed the LxLxL-type EAR motif (Fig 3).

The maize TIFY family members cluster into six distinct clades
To understand the evolutionary relationship of TIFY genes in maize, a phylogenetic tree of ZmTIFY proteins was created by the software Mega7 using the maximum likelihood method. The phylogenetic tree showed that all the TIFY proteins in maize clustered into six clades ( Fig  4). Interestingly, ZmTIFY1 and 5 clustered with ZmJAZ8, 9, 13, 17, 23, 26, 32 in clade II (Fig  4) while ZmTIFY2 and 3 clustered with ZmJAZ 4, 5, 7, 14, 15, 16, 19, 25 and 37 in clade IV (Fig 4) and the four ZmZML proteins clustered into separate clades (I, II, II, and VI) (Fig 4). Comparison of maize JAZ proteins with orthologues from Arabidopsis and sorghum found seven clear groups formed with Arabidopsis JAZ proteins clustered into four groups: G1, G3, G4, G5 and G7 while JAZ proteins in maize and sorghum clustered into 6 groups: G2 to G7 (S1 Fig).

All maize JAZ promoters contain JA responsive regulatory elements
Promoter sequences of the ZmJAZ genes were analyzed via the PlantCARE database to identify cis-regulatory elements in the1.5kb promoter segment upstream of their start codons. Attention was given to elements relevant to hormone and stresses responses (Fig 5; S2 Table). Those elements included: (1) the ABRE motif, involved in abscisic acid (ABA) responses; (2) MBS, a MYB transcriptional factor binding site involved in drought tolerance; (3) MYC, a transcriptional factor of JA responsive genes; (4) the CGTCA-and TGACG-motifs, involved in methyl-JA acid (MeJA) responses; (5) the AuxRR-core, TGA-element, and AuxRE, the auxin-responsive elements; and, (6) the GARE-motif and TATC-box, that serve as gibberellin (GA) -responsive elements.
All putative ZmJAZ promoters contained at least two different regulatory elements (Fig 5) with some, such as ZmJAZ9, possessing all six elements of the analysis. All ZmJAZ promoters contained either MYC-binding site or CGTCA/TGACG-motif for JA and MeJA responses, respetively (Fig 5). Most ZmJAZ promoters contained one to several ABRE motifs are involved in abscisic acid (ABA) responsiveness (Fig 5).

Most ZmJAZ genes have non-specific expression across maize tissues under basal conditions
To gain insight into tissue-specific expression of the maize TIFY genes, publicly available transcriptomes of 79 different maize tissues and organs [46] were mined. Of the 43 genes identified in our study, 34 TIFY genes were found transcribed at basal levels in at least one of the available tissue types (Fig 6) and several patterns emerged.
Apart from ZmJAZ32 and 36, all ZmJAZ genes tested were found to be transiently induced by wounding . ZmJAZ5, 6, 15, and 17 displayed a rapid, but short, increase in wound-inducible expression; induction of these genes was detected at 1 and 3 h post-wounding, but subsided by 6 h. Wounding induced expression of ZmJAZ3, 8,9,12,18,25,31, and 33 as early at 1 h following treatment, but their induction persisted for the duration of the time-course. ZmJAZ13, 20, and 23 appear to be late wound-induced genes and it is likely their peak of their expression was not captured within the time-points tested (Fig 7A).
Mechanical damage induces production of JA and subsequent JA-responsive gene expression. To test the contribution of JA in wound-inducibility of ZmJAZ genes, maize seedlings were chemically treated with JA. With the exception of ZmJAZ15, 17, and 20, most maize JAZ genes were found to be JA-inducible. Expression of ZmJAZ8, 11,12,18,25,31,32, and 33 were induced as early as 6 h and persisted for at least 24 h after treatment. Transcription of ZmJAZ5, 6, and 9 was detected at only 24 hours post wounding. ZmJAZ15 and ZmJAZ20 were constitutively expressed in the leaves and unresponsive to JA treatment and remarkably, ZmJAZ17, the most highly expressed gene in the leaves (Fig 6), was the only ZmJAZ observed to be repressed by JA treatment (Fig 7B).
Comparison of expression patterns between mechanically wounded and JA treated plants found that the majority of ZmJAZ genes induced by mechanical damage were also JA-responsive. Interestingly, several genes responded differently to JA treatment and wounding in the time-points measured. Wounding, but not JA-treatment, induced ZmJAZ15 and 20, however the opposite was observed for ZmJAZ32 (Fig 7).

Discussion
In recent years, advances in sequencing technologies have enabled substantial improvements to the maize reference genome providing a more accurate representation of the genomic composition and subsequent gene models. In this study, we used updated B73 reference genome AGPv4 to identify and categorize 47 TIFY family genes, named ZmZIM1 to ZmZIM47 (Table 1). This work augments the existing literature with over 40% more maize TIFY members, compared with previous studies that identified only up to 30 isoforms [15,[40][41][42]. More specificially, our analyisis uncovered five, four, and 38 genes to the TIFY, ZML, and JAZ subfamilies, respectively and were named accordingly. No PDD subfamily members were identified during this process, consistent with what is currently understood from other monocot species [15,41]. Compared with other grasses, the maize genome encodes more than twice the number of predicted TIFY genes than Brachypodium [36], rice [32], or sorghum [15], and thus far only wheat is known to contain more with 47 identified.
Maize arose from a hybridization of two ancestral species that produced an allotetraploid approximately 14 million years ago and soon after underwent diploidization [52] resulting in a segmental alleotetraploid [53]. The closest crop species relative to maize is sorghum (Sorghum bicolor) which diverged from one of the maize ancestors around the same time of the maize hybridization event. Using the sorghum genome as a guide, segments from the two maize subgenomes (maize1 and maize2) can be differentiated where each subgenome is orthologous to the sorghum genome [49]. In our analysis, 32 and 14 TIFY genes were identified on the maize1 and maize2 genomes, respectively (Fig 1). This observation agrees with the finding that in modern maize inbred lines, maize2 has exhibited significantly more gene loss compared to maize1 [49].
Prior to the discovery of JAZ proteins [10,12,33], the functional annotation of the plant-specific TIFY family was unclear [13]. [15] analyzed the origin and evolution of the TIFY genes and organized them into four subfamilies: ZML, TIFY, PPD and JAZ where the latter can account for 60-80% of the TIFY genes in a species and undeniably are the best understood. In Arabidopsis, JAZ proteins are transcriptional repressors for JA-mediated response [10][11][12]. During JA signaling, JA-Ile serves as a ligand to promote the formation of a SCF COI1 -JA-Ile-JAZ complex in which JAZ proteins are ubiquitinated and subsequently degraded by the 26S proteasome [10,11].
JAZ proteins contain a TIFY and Jas domain in their N-and C-terminus, respectively, and both domains are required during JA signal transduction. The TIFY domain is necessary for homo-and heteromeric dimerization between the TIFY family members [50] and for JAZ-NIN-JA-TPL interaction and the Jas domain is required for the formation of the COI1-JAZ co-receptor complex [51]. In maize, five TIFY subfamily proteins (ZmTIFY1, 2, 3, 4, and 5) contain solely a TIFY domain (S1 Table; Fig 3), however they are highly similar with typical ZmJAZ proteins and cluster with them during phylogenetic analysis (Fig 4). These observations suggest that the maize TIFY subfamily are comprised of JAZ proteins that have lost their Jas domains during the evolutionary process. In contrast, several ZmJAZ proteins possessed normal Jas domains but either lacked (ZmJAZ 29, 30, 31, 32, and 36) or have incomplete (ZmJAZ25 and ZML4) TIFY domains (S1 Table; Fig 3), which likely results in the inability to dimerize normally with other JAZ proteins and the subsequent loss of function as transcriptional repressors.
Insect herbivory or mechanical damage rapidly induce expression of JAZ genes in Arabidopsis, and functional analysis with aos and coi1 mutant lines showed that both JA biosynthesis and perception are required in this process [12,30]. In this study, we found that 14 ZmJAZ genes (ZmJAZ3, 5,6,8,9,11,12,13,18,20,23,25,31, and 33) are induced by either mechanical wound, exogenous JA application, or both treatments (Fig 7). These results provide pharmacological evidence that JAZ genes from diverse plant species respond by similar cues resulting in similar defensive functions in both monocots and dicots.
Promoter analysis provides insights into the regulation of genes to elucidate their physiological functions. Here, we examined the ZmJAZ promoters for six cis-regulatory elements involved in defense and hormone responses. ABA facilitates stomatal closure in response to abiotic stress such as during drought conditions [54], while auxin signaling regulates tolerance to diverse stresses [55]. SA, JA, and ET are best understood for their roles in plant defense to diverse biotic and abiotic stresses. Here, they activate transcriptional reprogramming to engage defense against various pathogens, pests, and abiotic stresses, such as wounding and salt [56]. JA and ET usually synergistically regulated plant development and tolerance to necrotrophic fungi [57]. GA is a major growth hormone and stress-induced growth reduction is associated with decreases in GA levels [58]. Our result revealed that ZmJAZ gene promoters contain several cis-regulatory elements related to plant hormone and stresses regulation. This is in agreement with a recent study that identified cis-elements associated with ABA, Auxin, MeJA, GA, and stress tolerances in promoters of wheat JAZ genes [43] and consistent with an increasing number of studies that have functionally characterize specific JAZ proteins in plant hormone regulation of defense responses against abiotic and biotic stresses in rice, tomato, maize, and poplar [32,33,42,59]. Thus, it is reasonable to expect that the maize JAZ proteins will emerge as potent mediates in crosstalk hormone signaling crosstalk during plant growth, development, or defense processes.