Identification and comparative genomics of OVATE Family Members from Gramineae uncovers sequence and structural diversity, evolutionary trends, and insights into functional features

doi:10.21203/rs.3.rs-5150792/v1

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Identification and comparative genomics of OVATE Family Members from Gramineae uncovers sequence and structural diversity, evolutionary trends, and insights into functional features

https://doi.org/10.21203/rs.3.rs-5150792/v1

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Ovate Family Proteins (OFPs) are plant-specific, transcriptional repressors characterized by the presence of an OVATE domain. The OFP gene family has been analysed only from a handful of species, and functionally characterized from even fewer plants. Members of Gramineae have been subject of several investigations, and now has complete genome sequence for several species. Such analyses revealed diversity in genome size, ploidy and chromosome number. However, there exists a gap in cataloguing the complete compendium of OFP family across Gramineae. In the present study, we therefore identified and catalogued homologs of OFPs across ten Gramineae members to analyse gene and protein structure and properties, evolutionary relationship, expression pattern, and predict interacting partners. A positive correlation was found between genome-size and OFP family size, with Triticumgenome harbouring the maximum number; most of the Gramineae OFPs are intronless. Comparative analysis revealed variation in gene sizes, protein physico-chemical properties and structures including motifs. Phylogenetic reconstruction reflected homolog-based clustering. Expression analysis in Oryza revealed spatio-temporal variation with maximum expression in reproductive tissues. Prediction of interactome showed homeobox domain containing proteins as major interacting partners. The study thus form foundation for future functional analysis of role of OFPs in regulating economically important traits.

Plant Molecular Biology and Genetics

Botany

Gramineae

OVATE Family Proteins

Rice

Transcriptional Repressor

Tightly regulated and coordinated gene expression patterns are critical to development, perception of environmental stimuli, and adaptive responses. Accurate and synchronized gene expressions are driven by complex regulatory networks, and involve activator and repressor complexes which act to fine-tune the transcript levels (Deckert and Struhl, 2001; Reynolds et al., 2013). Although significant advances have been made towards characterizing transcriptional activators, knowledge about identity and role of transcriptional repressors are scanty.

OVATE family proteins (OFPs) are plant-specific transcriptional repressors characterized by the presence of an OVATE domain of approximately 70 amino acids at the C-terminal end. OFPs also harbor a bi-partite NLS, and two putative Von-Willebrand factor type C (VWFC) which are vital for protein–protein interaction (Liu et al., 2002; Wang et al., 2016, 2011). Even though OFPs have been shown to be present across land plants, extensive analysis of its role in various developmental and adaptive pathways, and functional diversity vis-a-vis their evolutionary divergence is lacking (Chahar et al., 2021; Dangwal and Das, 2018; Liu et al., 2014). Experimental evidence of role of a few OFPs has been demonstrated only in A. thaliana and O. sativa, with information about rest of the members being limited or not available. Studies have examined the role of OFPs in growth and development in only a handful of plants, primarily Arabidopsis thaliana (Hackbusch et al., 2005; Wang et al., 2011; Zhang et al., 2016), O. sativa (Ma et al., 2017; Schmitz et al., 2015; Wang et al., 2007; Yang et al., 2018, 2016; Yu et al., 2015), Solanum lycopersicum (Huang et al., 2013; Liu et al., 2002), Malus domestica (Li et al., 2019) and Musa acuminata (Zhang et al., 2020).

Inspite of the large variation in genome size, content, chromosome number and ploidy levels, comparative mapping has revealed remarkable conservation and collinearity of genes and genetic markers amongst Gramineae genomes (Feuillet and Keller B., 2002). The conserved nature of gene structure and order has allowed O. sativa (rice) to be developed as a model system for structural and functional genomics of grasses (Kumar et al., 2009). Rice is also the primary source of staple carbohydrate to nearly 50% of world’s rice accessions to be sequenced and analysis reveals high levels of intra-specific genetic diversity. More so, intra-specific genetic diversity is also known to play a crucial role in nutritional diversity. (Reddy Lachagari et al., 2019; Sun et al., 2017). The availability of whole genome sequences has opened new windows for the identification and characterization of genes and gene family (Nuruzzaman et al., 2010). Apart from O. sativa, genome of several other monocot species such as O. coarcata (Mondal et al., 2018), O. rufipogon (Li et al., 2020), Zea mays (Schnable et al., 2009), Brachypodium distachyon (Vogel et al., 2010), Triticum aestivum (Brenchley et al., 2012), Pennisetum glaucum (Varshney et al., 2017), Sorghum bicolor (Paterson et al., 2009), Hordeum vulgare (Beier et al., 2017), Aegiolops tauschii (Luo et al., 2017) and Setaria italica (Zhang et al., 2011) are now available for analysis.

Gene families expand through local, segmental, and whole genome duplications generating paralogous copies. Subsequent selection pressures cause regulatory and functional diversification through sub-functionalization, neo-functionalization and pseudogenization leading to divergence (Bornberg-Bauer and Albà, 2013; Cheng et al., 2018; Gu et al., 2002; Hughes, 2002; Lespinet et al., 2002; Zhang, 2003).

The presence and occurrence of protein domains has been used as the primary criteria to classify and organize gene and protein families (Dangwal et al., 2014, 2013; Marsh and Teichmann, 2010; Mishra et al., 2018). Inspite of progress made, there exists a significant gap in complete cataloguing of homologs of OFPs in members of Gramineae, including understanding about genomic organization, evolution, and putative function/s. Availability of genome sequence thus will not only permit the analysis of synteny, but also the detailed analysis of molecular diversity and evolution of the OFP gene families. Fragmentary information is available about OFPs from grasses in general and O. sativa in particular. In the recent past, a few scattered reports on functional characterization of selected OFPs from O. sativa has appeared including on subcellular localization (Yu et al., 2015), and in brassinosteroid signaling (Xiao et al., 2017; Yang et al., 2018, 2016).

In the present study, we therefore identified and catalogued the entire compendium of homologs of OFPs across nine different species from monocot using rice as a model template to understand their organization, structural and evolutionary affiliations, and inferred functional importance and relatedness. To the best of our knowledge, this is the first comprehensive study on comparative analysis of OFPs in grasses and the findings of the present study will provide the necessary framework to understand evolutionary processes underlying sequence and structural diversity, basis of functional diversification, and form the basis for functional characterization.

2.1 Identification of Ovate Family Proteins (OFPs), structural organization and chromosomal localization

OVATE family protein members from Oryza sativa (Os) were searched in TIGR database (http://rice.plantbiology.msu.edu/) and in the genomes of Brachypodium distachyon (Bd), Brachypodium stacei (Bs), Panicum hallii (Ph), Panicum virgatum (Pv), Setaria italica (Si), Setaria viridis (Sv), Sorghum bicolor (Sb) from phytozome database (version 12) (https://phytozome.jgi.doe.gov/pz/portal.html). Zea mays (Zm) and Triticum aestivum (Ta) OFP candidates were searched from Ensembl Plants dataset (http://plants.ensembl.org/Multi/Tools/Blast). Protein sequences of all AtOFPs were used as query to identify homologs from genomes of selected grass species using BLOSUM62 algorithm in BLASTP with expected threshold value = 1e^− 1 in Phytozome as well as in Ensemble plants database and 1e^− 5 in TIGR database keeping “max number of alignments report = 10”, and “filter = none” as default parameters. Additional searches were performed using the terms “OVATE domain” and “OFP” for keyword search. Output lists were manually scrutinized and high scoring pairs (HSPs) were identified, and redundant entities were removed. Genomic and transcript sequences of all searched OFPs were retrieved and exon-intron organization were investigated with Gene Structure Display Server 2.0 (GSDS, http://gsds.cbi.pku.edu.cn/). For chromosomal localization of OsOFPs, MSU IDs were converted to RAP ID through RAP-DB. (https://rapdb.dna.affrc.go.jp/tools/converter/run). These IDs were then mapped through MapTool in Oryzabase (http://viewer.shigen.info/oryzavw/maptool/MapTool.do). Chromosomal location of OFP members were identified from the Phytozome and Ensembl Plants database and plotted through MapChart using genome coordinates (Voorrips, 2002). NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), EMBL SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.sanger.ac.uk) databases were used for analysis of conserved domain sequences. Identification and analysis of motifs were performed using Multiple EM for Motif Elicitation (MEME) (http://meme-suite.org/tools/meme) with size range between six and fifty amino acids. Protein subcellular localization was studied through CELLO (http://cello.life.nctu.edu.tw/), LOCALIZER (http://localizer.csiro.au/), WoLF PSORT (https://wolfpsort.hgc.jp/), DeepLoc-1.0 (http://www.cbs.dtu.dk/services/DeepLoc/) and ESLPred2 (http://crdd.osdd.net/raghava/eslpred2/).

2.2 Physical and biochemical properties of OFPs

Molecular weight of each OFPs and their isoelectric points were computed at ExPASy server (http://web.expasy.org/compute_pi/). Protein disorderness and hydropathy scores were calculated through RAPID (http://biomine.cs.vcu.edu/servers/RAPID/) and GRAVY (http://www.bioinformatics.org/sms2/protein_gravy.html) software according to the method described earlier (Shekhar et al., 2016). Conserved OVATE domains were analyzed for their putative tertiary structure through Phyre2 protein-modeling server (www.sbg.bio.ic.ac.uk/*phyre2).

2.3 Reconstruction of evolutionary relatedness and synteny analysis

Homologs of OFPs proteins identified from selected grass species were aligned using MAFFT tool and the output file was employed to reconstruct phylogeny by Bayesian method (BEAST) using Cipres Science Gateway (Miller et al., 2010). The tree was visualized and analysed using Figtree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). To understand the ancestry of each domain, character-state matrix was generated on the basis of their presence or absence. These characters were traced on the phylogenetic tree through Mesquite software for character state reconstruction (Maddison and Maddison, 2019). To study synteny, genomic regions surrounding OsOFPs were analysis at Plant Genome Duplication Database (PGDD) (http://www.chibba.agtec.uga.edu/duplication/) using Locus search tool. Paralogs and orthologs were visualized through ClicO FS (http://103.47.253.210:3000/projects?demo=1).

2.4 Plant growth, maintenance and tissue sampling

Seeds of Oryza sativa L. subspecies indica var. IR64 were germinated in 30-cm diameter pots (4–6 seedlings per pot) containing mixture of soil and peat (3:1) and grown under field condition following recommended agronomic practices. Tissue samples were harvested at eleven different stages (14-days old seedling, root of seedlings, young leaves, flag leaf, early panicle (0-10mm), mature panicle, leaves from mature panicle stage, leaves from young seedling stage, young seed, leaves from mature seed stage; mature seeds) and immediately frozen in liquid nitrogen and stored at -80ºC until processing. At least three biological and three technical replicates were used for experimentation.

2.5 Expression analysis of rice OFP members

Total RNA from 14-days old seedling, root of seedlings, young leaves, flag leaf, early panicle (0–10 mm), mature panicle, leaves from mature panicle stage, leaves from young seedling stage, and leaves from mature seed stage was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer instructions and lithium chloride method was used in case of young seed and mature seeds as described earlier (Bandyopadhyay et al., 2012). After RNase-Free DNase I (Qiagen) treatment, RNA was quantified and integrity was checked. 1 µg of total purified RNA was used to prepare cDNA with SuperScript® VILO™ cDNA Synthesis Kit as per manufacturer’s recommendation (Invitrogen). Real-time RT-PCR primers (Supplementary Table S1) were designed using Primer 3 software (Koressaar and Remm, 2007). Steady-state transcript abundance of each OsOFPs was quantified using SYBRGreen® PCR Kit (Applied Biosystems, USA). Mean Ct values were then used for quantification of relative abundance (delta-delta Ct; 2^–∆∆Ct) as per Livak and Schmittgen (2001) Transcript levels of rice tubulin was used as internal control (Jain et al., 2006).

To analyze the expression profile of OsOFPs, RNA-seq data was retrieved from TIGR database and analysed (http://rice.plantbiology.msu.edu/) for each OFPs in 20 Day leaves (SRX100741), emerging (Post-emergence inflorescence; SRX100743) and early inflorescence (Pre-emergence inflorescence; SRX100745), anther (SRX100746), pistil (SRX100747), 2 stages of seed [5 Days After Pollination (5 DAP; SRX100749) and 10 Days After Pollination (10 DAP; SRX100755)], embryo (SRX100753), endosperm (SRX100754), shoots (SRR042529) and seedling of four-leaf stage (SRX016110). Spatiotemporal transcript data of OsOFPs were used to generate the heatmap through Heatmapper (http://www.heatmapper.ca/expression/) (Babicki et al., 2016).

2.6 In-silico protein-protein interaction

Putative protein-protein interactions were identified using keyword search at Predicted Rice Interactome Network (PRIN) database (http://bis.zju.edu.cn/prin/) (Gu et al., 2011), and visualized in Cytoscape (version 3.4.0) (Maere et al., 2005).

3.1 Identification of Ovate Family Proteins

Amino acid sequences of Arabidopsis OFPs (AtOFPs) were used as a query to perform BLASTP and 31 homologs of OFPs were identified from the Oryza sativa genome at TIGR database (OsOFPs 1–31). We also identified homologs of OFPs from the genomes of Brachypodium distachyon (BdOFP; 1–32), Brachypodium stacei (BsOFP; 1–31), Panicum hallii (PhOFP; 1–30), Panicum virgatum (PvOFP; 1–58), Setaria italica (SiOFP; 1–34), Setaria viridis (SvOFP; 1–34), Sorghum bicolor (SbOFP; 1–37) using Phytozome database and OFPs for Triticum aestivum (TaOFP; 1-105) and Zea mays (ZmOFP; 1–43) from Ensembl Plants database (Supplementary Table 2). A comparison of the genome size, C-value with the size of the OFP family across the members of Gramineae showed a positive correlation (r = 0.95289). maximum number of OFPs (105) were found in T. aestivum a hexaploid with a genome size of ~ 17 Gb, and the lowest in the diploid P. hallii with a genome size of 487 Mb (Fig. 1).

We attempted to examine intron gain-loss across monocots and lower plants by including data from our earlier study (Fig. 1) (Dangwal and Das, 2018). The analysis revealed that even though monocots have significantly higher number of genes in the OFP family, the number of introns remained nearly constant signifying loss of introns during transition from bryophytes to monocots (Fig. 1).

3.2 Structural characteristics of OFPs

To understand the miscellany at the genomic and protein levels in OFPs across the members of Gramineae, we examined the structural attributes of different OFPs. The size of the 31 OsOFPs ranged from 432–1786 bps with a 4.1-fold variation between the two extremes; homologs of OFPs from Z. mays genome showed minimal variance and ranged between 1752–2560 bps (~ 1.5-fold), whereas those from P. virgatum exhibited the maximum variance from 279-11398 bps (~ 41-fold). Overall, the variation in gene sizes within the OFP family in other members of Gramineae ranged from 3.2-fold in TaOFPs (1524–4852 bps) and BdOFPs (729–2338); ~5-fold in SiOFPs (690–3432 bps); ~5.4-fold in SvOFPs (780–4214 bps); 9.5-fold in PhOFPs (606–5769 bps); 12.4-fold in SbOFPs (473–5907 bps) and 15.1-fold in BsOFPs, (684-10352 bps) (Supplementary Table S1).

We also examined the size of transcript and CDS of OFPs across the members of Gramineae. In nearly all members of OFP in O. sativa (except LOC_Os04g33870), the size of gene and transcript was found to be similar indicating the absence of introns; the predicted CDS was smaller than the transcript implying presence of UTR (Fig. 2A; Supplementary Table S2). Similar observations were noted in rest of the species, except in Z. mays and T. aestivum where the difference in sizes of genes, transcript and CDS in a number of OFPs suggest presence of introns and UTRs (Supplementary Table S2).

To understand the conservation and divergence in structural features of OFPs across members of Gramineae, protein sequences were analyzed using SMART and MEME-suites. The analyses revealed the presence of OVATE domain at the C-terminal (Fig. 2B; Supplementary Table S2). DNA binding domain (DBD) was detected in three OFPs each in O. sativa (LOC_Os01g60810, LOC_Os05g25910 and LOC_Os05g39950), B. distachyon (Bd2g22310, Bd2g53610 and Bd3g28000), B. stacei (Bs01G104000, Bs03G140600 and Bs08G085500), P. hallii (Ph.C02605, Ph.C03367 and Ph.E01138), S. italica (Si.3G205300, Si.3G286300 and Si.5G363400), S. virdis (Sv.3G210200, Sv.3G294300 and Sv.5G368800) and S. bicolor (Sb.003G339100, Sb.009G095800 and Sb.009G173400); two OFPs in P. virgatum (Pv.Ca01960 and Pv.J01009), four OFPs in Z. mays (Zm00001d043046, Zm00001d038473, Zm00001d009993 and Zm00001d012498) and eight OFPs in T. aestivum (TaCS1A02G300100, TaCS1D02G299500, TaCS2A02G392200, TaCS2B02G410500, TaCS2D02G390300, TaCS3A02G345100, TaCS3B02G377200 and TaCS3D02G339100) (Supplementary Table S2).

Apart from the DBD, two repeat variants ranging from 12–42 amino acids and annotated as internal repeat domain (RPT) located upstream of OVATE domain were detected in selected few homologs of OFP across few species. RPT was detected in one OFP each of O. sativa (LOC_Os04g48830), P. halli (Ph.G02026), P. virgatum (Pv.Ga00975) and S.italica (Si.7G208400); and four OFPs in T. aestivum (TaCS2A02G106200, TaCS2A02G396600, TaCS2D02G394400 and TaCS5B02G125500; Supplementary Table S2).

In addition to DBD and RPT domain, two conserved motifs, each of 29 amino acids, were detected towards the C-terminal of the protein, overlapping with the OVATE domain. Homologs of nearly all OFPs across most of the species showed the presence of both the motifs. Some of the OFPs harbours either the first (Pv.J30311) or second (Bd2g24002, Bd3g27170, Bs03G131900, Pv.Db02258, Pv.Ib02475, Pv.J36184, Sb.003G262000 and TaCS5D02G070300) motif. One OFP from Z. mays (Zm00001d038284) and five from T. aestivum (TaCS2A02G106200, TaCS2B02G123200, TaCS2D02G106000, TaCS4A02G130500 and TaCS4B02G173800) lack both the motifs (Supplementary Table S2).

3.3 Putative functional characterization of OFPs

An in-silico analysis was performed, first employing CELLO (http://cello.life.nctu.edu.tw/) tool using “eukaryote” as the option, followed by validation using LOCALIZER (http://localizer.csiro.au/), WoLF PSORT (https://wolfpsort.hgc.jp/), DeepLoc-1.0 (http://www.cbs.dtu.dk/services/DeepLoc/) and ESLPred2 (http://crdd.osdd.net/raghava/eslpred2/) to predict sub-cellular localization of OFP proteins. Majority of the members of OFP family across all members of Gramineae were found to be nuclear localized with a few proteins predicted to be localized in chloroplast, mitochondria, plasma membrane and extra cellular matrix (Fig. 3A; Supplementary Table S2).

In order to understand the sequence-structure relationship, we predicted the tertiary structure using the amino acid sequence of the OVATE domain employing the PHYRE2 tool (Kelley et al., 2015). The OVATE domains derived from majority of the OFPs across the species showed homology to one of the following three structures - Programmed cell death 4 protein (PDCD4) C-terminal MA-3 domain, CO-type nitrile hydratase alpha subunit and MIF4G domain-like. Among the various species, OVATE domain of 29 O. sativa OFPs share homology with PDCD4 C-terminal ma-3 domain, and that of two OFPs with co-type nitrile hydratase alpha subunit; none of the OVATE-domains showed structural similarity with MIF4G domain-like (Fig. 3B; Supplementary Table S2). Most of the OFPs from other species display similarity with PDCD4 C-terminal MA-3 domain; OVATE domains from only four OFP homologs- Bd2g07543 from Brachypodium distachyon, Si.3G130900 from Setaria italica, Sv.3G132300 from Setaria viridis and Sb.001G523500 from Sorghum bicolor showed homology with MIF4G domain-like. Out of a total of 435 OFP homologs across fourteen species analyzed, OVATE domain from only 47 (ca. 11%) OFPs shared homology with co-type nitrile hydratase alpha subunit. Apart from homology to the three predominant structural classes, OVATE domains of a few OFPs showed homology to other structural folds such as to alpha-hemoglobin stabilizing protein (AHSP) fold (Si9G558100); phospho ribosyl formyl glycinamidine synthase (Zm00001d034973); hemoglobin-binding protease (hbp) (Si.5G128600); RNA polymerase-binding protein (RPBP; Pv.Db02258); synaptonemal complex central element protein 3 (Pv.Ib02475). (Supplementary Table S2).

3.4 Physico-chemical properties of OFPs

The OFPs across the grass species were analyzed for their physical and biochemical properties by predicting molecular mass (KDa), pI, GRAVY values and intrinsic disorderness. An analysis of the MW revealed that MW ranged from as small as 63 KDa (Pv.J36184) to as large as 600 KDa (Sb.003G339100). Out of 435 OFPs analyzed, 228 (52%) fall in the size range of 201–300 KDa, followed by 145 OFPs that fall into size range of 301–400 kDa. A species-wise analysis revealed that in O. sativa LOC_Os04g37510 has lowest MW (~ 156 kDa) and LOC_Os05g39950 highest (~ 523 kDa) (Fig. 4A). In B. distachyon, the lowest and highest MW are 162 kDa (Bd3g27170) and 416 kDa (Bd2g07543); in B. stacei, MW ranges between 219 kDa (Bs03G131900) to 478 kDa (Bs08G085500); in P. hallii, MW ranges between 206 kDa (Ph.E04218) to 407 (Ph.C00473) kDa; between 63 kDa (Pv.J36184) to 409 (Pv.Ga00975) kDa in P. virgatum; in S. italica, OFP MW ranges from ~ 206 kDa (Si.9G533600) and ~ 440 kDa (Si.7G208400) KDa; and, between 206 kDa (Sv.9G537900) to 486 kDa (Sv.5G010900) kDa in S. viridis. The MW of OFPs ranged between ~ 154 kDa (Sb.003G262000) to 601 kDa (Sb.003G339100) in S. bicolor; between 187 kDa (Zm00001d027739) and 456 kDa (Zm00001d044167) kDa Z. mays; and, between 117 kDa (TaCS5D02G068300) to 419 kDa (TaCSU02G205700) in T. aestivum (Fig. 4A).

Prediction of isoelectric point (pI) of the OFPs revealed a range of 4.19 to 11.69; nearly 65% of all OFPs (285 out of 435) have pI values > 8, and 31% (136 out of 435) have < 8. Similar trend was seen across all the genomes wherein majority of the OFP proteins are likely to have pI in the alkaline range (8–14). Analysis of pI values across the various genomes showed that OFP proteins with pI in the alkaline range was between 58% (P. virgatum) to 72% (Z. mays); and in the acidic range (4–7) was between 26% (S. virdis) to 38% (B. stacei) (Supplementary Table S2).

Grand average of hydropathicity index (GRAVY) values, a measure of hydropathicity and hydrophilicity, were computed for the putative OFP proteins, analyzed and compared. All the OFPs were found to be hydrophilic as their GRAVY values fall the negative range, between − 0.94 to − 0.001. GRAVY values of OsOFPs were in the range of − 0.823 (minimum; LOC_Os01g40970) to − 0.001 (maximum; LOC_Os10g29610). The GRAVY values of most OFPs fall in the range of -0.5 to -0.7 (Fig. 4C). We also analyzed the native disorder of OFPs via regression-based accurate predictor of intrinsic disorder (RAPID), a measure of polarity and native disorder and a comparative analysis was performed. Native disorder of OsOFPs varies approximately between 19–48%. LOC_Os04g58820 and LOC_Os01g53160 exhibited lowest and highest percentage of native disorderness, respectively. Other grasses showed ~ 12–66% of disorderness (Fig. 4D).

3.5 Distribution and chromosomal location

In order to understand the organizational pattern, and evolutionary history of OFP gene family in rice, their position on chromosomes was plotted using Maptool (http://viewer.shigen.info/oryzavw/maptool/MapTool.do). The haploid chromosome number of O. sativa is n = 12, and the genome contains 31 OFPs. Chromosome 1 was found to harbor as many as eight OFP genes (maximum) whereas chromosomes 2, 7 and 8 contain only one OFP each. Chromosomes 6 and 9 do not contain any OFP gene. A pair of OFPs were located each on chromosome 11 (LOC_Os11g05770 and LOC_Os11g05780) and 12 (LOC_Os12g06150 and LOC_Os12g06160). An analysis of the chromosomal coordinates (LOC_Os11g05770-Chr11: 2659043–2660499; LOC_Os11g05780-Chr11: 2665214–2666529; LOC_Os12g06150-Chr12: 2898691–2900071; and, LOC_Os12g06160-Chr12: 2911273–2912589) suggests that members in each of the pair of OsOFPs are tandemly organized (Fig. 5; Supplementary Table S2). An analysis of the phylogenetic tree comprising of O. sativa OFPs revealed that LOC_Os11g05770 and LOC_Os12g06150 are part of the same clade implying their paralogous nature. A similar relationship was also established for LOC_Os11g05780 and LOC_Os12g06160. The 32 OFP genes of B. distachyon were found to be distributed on all the five chromosomes with a maximum of 13 OFP genes on chromosome 2, and only three on chromosome 4 (Supplementary Fig. 1a). In Brachypodium stacei, 31 OFP genes were distributed on eight out of ten chromosomes; chromosomes 7 and 10 do not contain any homolog of OFP. B. stacei chromosome 1 harbours as many as 10, the maximum number, whereas chromosomes 4 and 6 have one OFP gene each (Supplementary Fig. 1a). The haploid chromosome number of P. hallii is n = 9, and the 30 OFP genes were found to be distributed on eight chromosomes, barring chromosome 4. The OFPs were found to be localized on top or bottom arms of chromosomes 3, 5, 7 and 9, with chromosome 5 harboring seven OFP genes (Supplementary Fig. 1a). The 58 OFPs of P. virgatum were distributed on fifteen out of eighteen chromosomes; three chromosomes- 2a, 4a and 8a do not contain any OFP gene. Chromosomes 5a and 5b contain maximum number of five OFP genes each. In S. italica (n = 9), OFPs were found to be distributed on eight scaffolds; nine out of 34 OFP genes were localized on scaffold 5, and eight on scaffold 3. Two scaffolds (Scf 1 and Scf6) harbor single OFP each; no OFP gene was found to be located on scaffold 4 (Supplementary Fig. 1a). S. viridis has 34 OFP genes and these were found distributed on eight out of nine chromosomes (Supplementary Fig. 1a). In S. bicolor (n = 10), 37 OFP genes were distributed on all ten chromosomes with chromosome 3 having the maximum numbers (10 genes) (Supplementary Fig. 1a). Similarly, the 43 OFP genes in Z.mays were distributed on nine out of ten chromosomes with chromosome 3 harbouring the maximum number (8 genes; Supplementary Fig. 1b). In T. aestivum 105 OFP genes were distributed on the 21 chromosomes as well as on the scaffold. Wheat chromosomes 2A, 2B, 2D, 3A, 3B, 3D, 4A, 4B and 4D harbored most of the TaOFPs (Supplementary Fig. 1b). A common feature across all the genomes surveyed was clustered localization of majority of OFP genes on chromosomes.

3.6 Evolutionary relationship

To understand the relationship of OFPs among members of Gramineae, an unrooted evolutionary tree was reconstructed by Bayesian method using BEAST through Cipres Science Gateway. Protein sequences of the 435 OFPs homologs from monocot were analyzed, along with 19 homologs of OFPs from Arabidopsis thaliana. Phylogenetic analysis was performed with both full-length protein sequence, as well as only the OVATE domain.

Reconstruction of the phylogeny revealed homolog-based clustering rather than species-specific clade differentiation using both full-length proteins sequence (Fig. 6) as well as based only on OVATE domains (data not shown). The sequence of 454 OFP proteins formed 12 distinct clades, with few homologs not part of any specific clade. Out of 12-subfamilies, the smallest sub-clade comprised of only five homologs from TaOFPs, four of which do not possess OVATE domain, except TaCS2A02G106000. Two AtOFPs (AtOFP19 / AT1G06923, and AtOFP16 /AT2G30395) which are characterized by partial OVATE-domain also grouped with this clade (Fig. 6). A comparative within- and between-clade analysis of sequences revealed presence of conserved and distinct amino acid motifs in OVATE domain present in different clades / branches of the phylogenetic tree (Fig. 6).

Out of 454 OFPs, nearly all OFPs were found to possess OVATE domain, and only few homologs harbor DNA binding domain and internal repeat 1 (RPT1) domain. We therefore also mapped the presence of these domains on the phylogenetic tree through ancestral state reconstruction. Ancestral state character reconstruction revealed that loss of OVATE domain, DNA binding domain, and internal repeats are independent events that occurred in a lineage-independent / homolog –independent manner OFPs (Fig. 7). The presence of DNA binding domain in 39 out of 454 OFPs indicated their independent gain during the evolution (Supplementary Fig. 2). Interestingly, a total of only six out of 454 OFPs possess two internal repeat (RPT1) domains and only two members possess a single repeat. Distributions of these repeats are suggestive of independent gain and loss of this during the course of evolution (Supplementary Fig. 3).

3.7 Duplication status of OsOFPs

Phylogenetic reconstruction allowed us to identify OFP homologs that are part of specific clades. In order to understand whether homologs that are part of sister clades arose as a result of segmental or local duplications, we analyzed the Plant Genome duplication database (PGDD) using OsOFPs. The Plant Genome duplication database (PGDD) was searched using the TIGR Locus IDs of the OsOFPs and we identified 18 pairs of OsOFP which are paralogs. Out of these, three OFPs from chromosome 1 showed duplicated status with 6 other OFPs. Different OFPs from chromosome 2, 3, 4, 5, 8 and 10 showed similar duplication events such as LOC_Os01g54570 showed single duplication with LOC_Os05g44090; few of these mapped on multiple duplications such as LOC_Os01g60810 exhibited duplication with LOC_Os03g21870 and LOC_Os05g39950 (Fig. 8). A cross-species examination of PGDD revealed that 22 protein pairs of Brachypodium showed high similarity with 14 OsOFPs, and 18 SbOFPs were similar to 15 OsOFPs.

3.8 Expression profiling of OsOFPs

To understand the possible role of OFPs during rice development, we analyzed the spatio-temporal expression pattern of all the 31 OsOFPs in eleven different stages of O. sativa IR64 grown under conditions as described in section 2.5; tissues were harvested from four vegetative and seven reproductive stages. Real-time qRT-PCR was performed with three biological and three technical replicates and transcript levels of rice tubulin was used as internal control. Expression of all the OsOFPs were detected in all developmental stages. LOC_Os01g12690 was found to be expressed at a lower level in all tissues except at mature panicle stage; similarly, transcript levels of LOC_Os01g40970, LOC_Os01g60810, LOC_Os02g45620, LOC_Os03g06350, LOC_Os04g58820, LOC_Os05g25910, LOC_Os05g36990, LOC_Os07g48150 and LOC_Os12g06150 were found to be higher at mature panicle stage. In contrast, transcript levels of LOC_Os01g43610, LOC_Os01g54570, LOC_Os01g64410, LOC_Os04g33870, LOC_Os04g37510, LOC_Os04g48830, LOC_Os05g12808, LOC_Os05g36970, LOC_Os05g44090, LOC_Os08g01190 and LOC_Os11g05770 were found to be high only during early stage of panicle development (Fig. 9A). Steady-state levels of OsOFPs LOC_Os01g53160 and LOC_Os03g21870 showed higher transcript abundance only in the young seed stage and found to be low in other developmental stages, whereas transcript levels of LOC_Os01g64430, LOC_Os03g03480, LOC_Os03g10150, LOC_Os05g39950, LOC_Os10g29610, LOC_Os11g05780 and LOC_Os12g06160 were upregulated at mature seed stage (Fig. 9A). LOC_Os10g38880 was the only OsOFPs which exhibited increased transcript in vegetative stage, particularly in roots (Fig. 9A).

Spatio-temporal expression pattern of all OsOFPs was also evaluated in 11 different vegetative and reproductive stages of rice based on in-silico analysis of RNA-Seq data available at TIGR. The expression profile, represented as heat-map, indicated that all the OsOFPs have relatively lower levels of transcript abundance in seedling stage (except LOC_Os01g43610, LOC_Os01g53160, LOC_Os02g45620) and in 20-day leaves; almost all OsOFPs, except LOC_01g12690, LOC_Os01g60810, LOC_Os05g39950, LOC_Os07g48150, LOC_Os10g29610, showed marginally higher level at early stages of inflorescence development (Fig. 9B). Some OsOFPs were found to be upregulated at specific-developmental stage. LOC_Os05g25910 showed high transcript accumulation specifically at emerging and early inflorescence while LOC_Os10g29610 showed highly induced expression at anther; LOC_Os05g39950 showed very high expression at endosperm and moderate at seed stage. Interestingly transcript of LOC_Os01g40970 could not be observed at any stage (Fig. 9B).

3.9 Putative interactome of OsOFPs

To predict the interacting partners of OsOFPs, a putative protein interactive network was generated. A total of 21 protein networks were identified from PRIN database for only 5 out of 31 OsOFPs. Analysis revealed that most of the networks showed putative interactions with homeobox protein knotted 1 or homeobox domain containing protein except LOC_Os05g25910 which displayed homodimerization. LOC_Os05g25910 and LOC_Os05g39950 showed highest number of interactive networks. Network analysis revealed that LOC_Os08g19650 which is a homeobox protein knotted-1 might interact with 4 out of 5 identified OsOFPs (Fig. 10).

Protein domains are spatially discrete segments, are considered as functional and/or structural units, and form basis of protein family classification. Being structurally and functionally important, protein domains are of considerable importance from an evolutionary perspective (i) because of their utility in identifying homologs present in different species; (ii) in investigations regarding protein evolution including proteins with permutation and combination of different domains; and (iii) understanding structure-function relationship. It is increasingly evident that functional conservation and divergence across protein orthologs and paralogs are governed by retention or loss of domains (Jakubec et al., 2018; Ponting and Russell, 2002). Novel domains emerge from existing repertoire of genes via different mechanisms, are responsible for structural and functional novelty, and emergence of new gene families (Dangwal et al., 2013; Malik et al., 2012; Marsh and Teichmann, 2010). Ovate Family Proteins (OFPs) are plant-specific, multigene family with a conserved OVATE domain. Homologs of OFP gene family have been reported spanning from early land plants lineages to monocots and dicots (Chahar et al., 2021; Dangwal and Das, 2018; Wang et al., 2016). Even though these have been reported in several plant genomes, limited information is available about their structural and functional diversity, particularly from an evolutionary perspective. Previously, we catalogued and examined genomic features and phylogeny of OFP homologs in the genomes of the early land plants represented by Marchantia, Physcomitrella, Selaginella, and Sphagnum, (Dangwal and Das, 2018), and across the members of Brassicaceae(Chahar et al., 2021). In the present study, we identified and analyzed homologs of OFPs in selected members of grasses including Oryza sativa (31 OsOFPs), Brachypodium distachyon (32 BdOFPs), Brachypodium stacei (31 BsOFPs), Panicum hallii (30 PhOFPs), Panicum virgatum (58 PvOFPs), Setaria italica (34 SiOFPs), Setaria viridis (34 SvOFPs), Sorghum bicolor (37 SbOFPs), Triticum aestivum (105 TaOFPs) and Zea mays (43 ZmOFPs) (Fig. 2A). No direct correlation was observed between the family size numbers of OFPs and the genome-size of the species (except T. aestivum) as had been observed in Arabidopsis (Hackbusch et al., 2005), tomato (Liu et al., 2002), pepper (Tsaballa et al., 2011), apple (Li et al., 2019), banana (Zhang et al., 2020) and early land plants (Dangwal and Das, 2018). The large family size in T. aestivum can be attributed to the hexaploid nature of the genome. A recent study in wheat reported that usually group of proteins harboring specific domain or tandem repeats are more prone to duplication events than that of those proteins in which that domain or tandem repeats is absent (Mishra et al., 2018). Different recent studies also revealed that the average number of OFPs in genome of dicots was lesser than that of the monocots (Hackbusch et al., 2005; Zhang et al., 2020).

Transcripts are considered as an operational unit of a genome (Gingeras, 2007). The correlation of the transcript with that of genome sizes vis-à-vis to the number of OFPs in rice and other grasses which exhibited almost a similar size of their transcript with that of their corresponding genomic size. Yet, it differs in case of LOC_Os04g33870 in rice and in Zea mays and wheat, suggesting the absence or loss of introns (Fig. 1, 2A).

Previous studies have uncovered several interesting features about gene families. For instance, a comparative analysis across early land plants lineage showed general trend towards the reduction in average length of transcript and protein from bryophytes to pteridophyte (Dangwal and Das, 2018), and that transcript size is inversely co-related to expression levels (Caldwell et al., 2015; Smith and Eyre-Walker, 2002). The presence of intron is believed to increase regulatory and transcriptional diversity in protein families (Chorev and Carmel, 2012; Meng et al., 2021; Rose, 2019), and analysis of OFPs across angiosperms such as A. thaliana, O. sativa, Malus sps, Musa sps have revealed these to be intron poor (Li et al., 2019; Yu et al., 2015; Zhang et al., 2020). The loss- and gain- / retention-of introns are an outcome of selection pressure; functionally significant introns have been retained and fixed because of Darwinian selection, whereas introns have been lost because of lack of positive selection (Belshaw and Bensasson, 2006). An earlier report in Arabidopsis and rice suggested that subsequent to a segmental duplication, the rate of intron loss is faster than the rate of intron gain (Lin et al., 2006). In a variety of plant lineages, low rates of intron gain have been reported, with intron losses being more frequent than the gains. For instance, the rate of intron loss in A. thaliana is ~ 12.6 times more than gain, whereas in rice, the loss is ~ 9.8 times higher than gain (Roy and Penny, 2007).

Domains and motifs in proteins are the key elements that contribute to structural and functional diversity of proteins (Forslund and Sonnhammer, 2012; Moore et al., 2008). These therefore are under negative / purifying selection which and do not permit accumulation of deleterious mutations that may negatively impact protein function (Camps et al., 2007; Neduva and Russell, 2005). Intra- and intergenomic rearrangements, and loss- and gain-of-domains are responsible for generating structural and functional diversity among protein homologs (Bornberg-Bauer and Albà, 2013; Kersting et al., 2012). Presence of DNA binding domain and RPT domain in OFPs in rice and other Gramineae members suggested their functional significance. Our analysis of distribution across plant lineage revealed independent events of gain and elimination of OVATE domain, DNA binding domain and internal repeats in some OFPs during the course of plant evolution (Fig. 2B).

OFPs are known to be a plant-specific transcriptional repressors; such repressors are known to have two functional domains - a DNA-binding domain and a repressor domain (Ohta et al., 2001). Although members of protein family, and transcriptional regulators with similar functions harbour similar domain/s, changes in specific amino acid residues within the domains have also been observed possibly to provide functional plasticity (Gonzalez, 2016). RPT domains are stretches of amino acids known to be present across species and participate in protein-protein interactions during several vital biological functions such as de-ubiquitination, and amelioration of stress responses, which are linked by involvement of protein-folding through ubiquitination-de-ubiquitination cycle and stress (Jaiswal et al., 2014; Kouranti et al., 2010; Sharma and Pandey, 2016). Presumably due to the higher degree of duplication in hexaploid wheat RPT-domains are proposed to have evolved through genetic recombination and intragenic tandem duplication; subsequent frequent duplications are responsible for diversity in both sequence and number of repeats even between orthologous genes, which possibly provides functional diversification to the RPT domain containing proteins (Andrade et al., 2001a; 2001b; Ma et al., 2017; Mishra et al., 2018; Sharma and Pandey, 2016).

Analysis of structure and function of protein/s is paramount in understanding the underlying molecular mechanisms (Vandromme et al., 1996). Subcellular localization of OFPs in rice and other grasses predicted through CELLO and LOCALIZER revealed that most of the OFPs were predicted to be localized in nucleus (Fig. 3A), which is consistent with their role as transcriptional repressors (Jian-ping et al., 2012; Reynolds et al., 2013; Wang et al., 2011; Withers et al., 2012; Yu et al., 2015). Several OFPs were organelle localized which likely is evidence of these as nuclear-encoded regulators of organellar transcription. Several proteins showed multiple localizations, and also outside the nucleus (Fig. 3A), because of their roles during developmental events which has also been reported earlier (Vandromme et al., 1996; Xin et al., 2021).

The 3-D structure of proteins is critical for interaction with other biomolecules, and functionality, and is influenced by altered protein sequence as a result of evolution. The OFP from homologs across members of gramineae exhibited similarity to PDCD4 C-terminal ma-3 domain, co-type nitrile hydratase alpha subunit and MIF4G domain-like (Fig. 3B; Supplementary Table 2), features which have also been reported from lower plants, indicating structural conservation (Dangwal and Das, 2018). PDCD4 protein is comprised of the two MA3 domains and recognized as a key module in translational initiation, and have been implicated in ethylene-mediated signaling and abiotic stress responses, particularly in the higher plants (Cheng et al., 2013; Lei et al., 2011). Co-type nitrile hydratases represent a group of metalloenzymes which are associated to catalyze the production of ammonia and organic acids using nitrile group containing compounds as substrate, and postulated to have been acquired by the eukaryotes through lateral gene transfer (Marron et al., 2012). Proteins containing MIF4G domain are reported to be similar to PDCD4 C-terminal MA3 domain, and are involved in translational initiation (Virgili et al., 2013). Besides these major classes, several unique domains were also observed such as, alpha-hemoglobin stabilizing protein (AHSP) in Setaria italica (Si.9G558100), phospho-ribosyl formyl glycinamidine synthase, hemoglobin-binding protease (hbp) (Si.5G128600); RNA-polymerase-binding protein RBPA (Pv.Db02258); and synaptonemal complex central element protein 3 (Pv.Ib02475) (Supplementary Table 2). The presence of different types of domains in homologs of OFP-gene family provides an opportunity to investigate relationship between structural and functional diversity in protein families; the origin of structural variants in the OFP gene family still remains to be investigated (Hackbusch et al., 2005; Liu et al., 2002; Schmitz et al., 2015).

Spatio-temporal expression analysis of the O. sativa OFP members across the eleven vegetative and reproductive stages using qRT-PCR, and in-silico analysis of the available microarray data of different vegetative and reproductive stages indicate their distinctive role in specific tissue and/or stage. Most of OFPs are differentially expressed, predominantly at reproductive stages including at early or mature panicle stage, or, young or mature seed stages; few of the members exhibited upregulation in vegetative stages (Fig. 9). Putative protein interactive network analysis of OFPs in O. sativa showed putative interactions with homeobox protein knotted 1 or homeobox domain containing protein which play key roles in regulating several traits of plant growth and development, hormone signaling (Fig. 10) (Chan et al., 1998; Jain et al., 2008; Kamiya et al., 2003; Sakamoto et al., 1999). Indirect evidences based on expression analysis of OFPs, and protein interaction network prediction indicating a coordinated mode of action between homologs of OFP and the homeobox protein KNOTTED1 needs to be experimentally validated.

Three-dimensional conformation of a protein is decided by its constituent amino acids which in turn determines molecular weight, isoelectric point (pI), hydrophobicity / hydrophilicity and influences structural variation (Aftabuddin and Kundu, 2007; Brown et al., 2010; Mishra et al., 2018). Several reports have investigated the role of mass and charge of protein to the spatio-temporal dynamics and interaction pattern; similarly, charge and polarity are indispensable for solubility of a protein, and ligand proximity (Mohanta et al., 2019; Xu et al., 2013). Indeed, a report also examined the correlation between pI, size and molecular mass of proteomes, and taxonomy and ecological niche of organisms (Kiraga et al., 2007). Analysis of native disorderness by RAPID exhibited a range of ~ 19–48% disorder in rice OFPs, while other grasses showed ~ 12–66% of disorderness (Fig. 4D). Concerning the composition and function of proteins, two different theories have developed. The 'structure-function model', which holds that a protein must have its natural three-dimensional structure under physiological conditions in order to function. The other theory is the recently developed "disorder-function model," which is based on proteins that carry out cellular tasks under physiological settings without achieving a stable three-dimensional structure (Trivedi and Nagarajaram, 2022). However, these proteins are implicated in several biological processes such as signaling, regulation of gene expression, cell-cycle regulation, amelioration of stress and many more regardless of their lack of any unique structure (He et al., 2009; Radivojac et al., 2007; Vucetic et al., 2007). Our earlier study of the OFPs from early land plants also showed that all these physicochemical parameters play a decisive role in species-specific diversity either individually or collectively, and are likely to be evolutionarily conserved (Dangwal and Das, 2018).

In conclusion, the present study provides a comprehensive cataloguing of the OFP gene family across the genome of ten-selected species of Gramineae. A thorough analyses of various features revealed variability in copy number, gene and protein structure, presence of introns, and domain composition suggesting probable functional divergence. Phylogenetic reconstruction indicates that the members of Gramineae inherited the entire OFP family from their last common ancestors as all species harbour atleast one copy of the homologs, and lineage-specific expansions were rare. Spatio-temporal expression analysis revealed differential transcript abundance across the developmental stages, with highest steady-state levels during reproductive stages in O. sativa. Prediction of interactome showed homeo-domain containing proteins as major interacting partners of majority of OsOFPs. The present study, to the best of our knowledge, thus provides a comprehensive collation of Gramineae OFPs with an exhaustive comparative analysis that will form the framework for evo-devo studies of multigene family proteins and understanding function and cross-species comparison, and identify candidates for functional analysis for crop improvement purposes.

Acknowledgments

The research has been supported and sponsored by the UGC under the UGC Dr. D.S. Kothari Post-Doctoral Fellowship and CSIR SRA Scientist’s Pool Scheme to MD [Award Letter: No.F.4-2/2006 (BSR)/BL/16-17/0032; 13(9116-A)/2020-Pool]. The award of JRF/SRF from CSIR to NC is gratefully acknowledged. SD would like to acknowledge Institute of Eminence (IoE), University of Delhi for financial assistance (Ref. No./IoE/2021/12/FRP), and University of Delhi for infrastructural support.

Competing Interest: The authors declare no conflict of interest

Author contributions

MD compiled the data, MD, NC and SD performed analysis. MD and SD drafted the Manuscript. All authors have read and approve of the manuscript.

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The authors declare no competing interests.

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Identification and comparative genomics of OVATE Family Members from Gramineae uncovers sequence and structural diversity, evolutionary trends, and insights into functional features

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1 Identification of Ovate Family Proteins (OFPs), structural organization and chromosomal localization

2.2 Physical and biochemical properties of OFPs

2.3 Reconstruction of evolutionary relatedness and synteny analysis

2.4 Plant growth, maintenance and tissue sampling

2.5 Expression analysis of rice OFP members

2.6 In-silico protein-protein interaction

3. Results

3.1 Identification of Ovate Family Proteins

3.2 Structural characteristics of OFPs

3.3 Putative functional characterization of OFPs

3.4 Physico-chemical properties of OFPs

3.5 Distribution and chromosomal location

3.6 Evolutionary relationship

3.7 Duplication status of OsOFPs

3.8 Expression profiling of OsOFPs

3.9 Putative interactome of OsOFPs

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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