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. 2015 Aug 11;10(8):e0134709. doi: 10.1371/journal.pone.0134709

Spt-Ada-Gcn5-Acetyltransferase (SAGA) Complex in Plants: Genome Wide Identification, Evolutionary Conservation and Functional Determination

Rakesh Srivastava 1,2, Krishan Mohan Rai 1, Bindu Pandey 1, Sudhir P Singh 3, Samir V Sawant 1,*
Editor: Swarup Kumar Parida4
PMCID: PMC4532415  PMID: 26263547

Abstract

The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses.

Introduction

The regulation of gene expression is accomplished by the coordinated action of multiple events to ensure a perfect synchrony of cellular activities from chromatin modification to mRNA formation [14]. Gene regulation in eukaryotes requires association of pre-initiation complex (PIC), transcription factors and activators at promoters [1, 5, 6]. One well-known mechanism for transcriptional activation suggests that activator proteins interact with promoter to recruit the components of transcriptional machineries and co-activators such as Transcription Factor II D (TFIID) complex, SAGA and mediator complexes [79]. The SAGA complex, a group of multi-protein complex, is important to induce the transcription of a subset of RNA polymerase II-dependent genes [1012]. Indeed, the SAGA complex is a perfect archetype for multi-subunit histone modifying complexes and co-activator which regulates transcription by RNA polymerase II [1315]. The first member of the SAGA complex family was isolated in budding yeast Saccharomyces cerevisiae [16]. The 1.8 megadalton S. cerevisiae SAGA complex is composed of 20 conserved proteins and contains different classes of transcriptional co-activator proteins such as SPT (Suppressor of Ty insertions), ADA (alteration/deficiency in activation), GCN5 (general control non-depressive), TAF (TBP-associated factors) proteins and DUBm (deubiquitylation module) [17]. These proteins are organized into different functional and structural sub-modules and thereby executing several cellular functions: nucleosomal histone acetyltransferase (HAT), histone deubiquitinylation, TATA-binding protein (TBP) binding and activator binding [10, 13, 18].

Interestingly, the SAGA complex is engaged in several transcription regulatory processes, for instance, facilitating recruitment of the RNA polymerase II, transcription elongation, promoting nucleosome eviction and replication-coupled nucleosome assembly [15, 1921]. In addition, the SAGA complex is associated with nuclear export of transcribed mRNA, co-transcriptional spliceosome assembly and transcriptional silencing at telomere region [2225]. Albeit, extensive evidences about the SAGA complex coding proteins in human, S. cerevisiae and other metazoan species are present, the knowledge about plants SAGA complex still needs to be elucidated. However, functions of few individual proteins of the SAGA complex are reported in plants, which have been shown to be involved in light signaling, stress response and histone modification [2631].

The present study aims at determining the genes encoding subunits of the SAGA complex across the plant species, mainly in the Arabidopsis thaliana and Oryza sativa, using in silico approaches, and exploring their potential roles in the gene regulation. We have highlighted the functional annotation, co-expression profiles and possible interactome among different proteins of the plant SAGA complex. To better understand the plant SAGA complex, we investigated its roles in regulating the light and stress-induced gene expressions in Arabidopsis.

Materials and Methods

Plant materials and treatment methods

Arabidopsis ecotype Columbia-0 (Col-0) seeds were grown on 0.5 x Murashige and Skoog (MS) medium, kept for 48 hr at 4°C, and then shifted for growth at 20°C ± 1 underneath white light (16 hr light/ 8 hr dark at 100–120 μmol·m−2·s−1). For the stress treatments, three-week-old Arabidopsis excised leaves were used. Excised leaves were placed either in 0.5 x MS medium (mock treatment) or in 0.5 x MS medium with 150 mM NaCl solution for 24 hr. For the heat/high temperature treatment, excised Arabidopsis Col-0 leaves were kept in 0.5 x MS medium and transferred into a 37°C incubator for 2 hr, whereas the control samples were kept at 22°C. The following mutants were used in the present study: gcn5‾ (Salk_030913c); sgf29b‾ (Salk_128344c); chr5‾ (Salk_020296c); tra1a‾ (Salk_087015c); taf12b‾ (Salk_132293c); sgf11‾ (Salk_088988c) and taf13‾ (Salk_024774c), which were acquired from the Arabidopsis Biological Resource Center [32].

Plant’s genome database search for identification of SAGA complex

National Centre of Biotechnology Information (NCBI); TAIR (The Arabidopsis Information Resource) and RAP (Rice Genome Annotation Project) databases were used for the screening of the SAGA complex in Arabidopsis, O. sativa and other plant genomes. Protein sequences of S. cerevisiae and human SAGA complex components (Table 1, S1 and S2 Tables) were used as queries to execute a BLASTP program against the protein sequences of Arabidopsis and O. sativa.

Table 1. SAGA complex classification in Arabidopsis and O. Sativa.

SAGA Subunits Yeast Human Arabidopsis thaliana Orzya sativa Functions
Name Locus Name Locus In plants Ref.
ADAs Ada1 ADA1 AtADA1a At2g14850 OsADA1a Os12g39090 -
AtADA1b At5g67410 OsADA1b Os03g55450 -
Ada2 ADA2b AtADA2b At4g16420 OsADA2b Os03g53960 Response to auxin and cytokinin; Pleiotropic effects in development; Abiotic stress [28, 99]
Ada3 ADA3 AtADA3 At4g29790 OsADA3 Os05g28300 -
Gcn5 (Ada4) GCN5 AtGCN5 At3g54610 OsGCN5 Os10g28040 HAT activity; Pleiotropic effects in development; Abiotic stress [28, 45, 99]
DUBm Ubp8 USP22 AtUBP22 At5g10790 OsUBP22 Os04g55360 -
Sgf11 ATXN7L3 AtSGF11 At5g58575 OsSGF11 Os05g28370 -
Sus1 ENY2 AtENY2 At3g27100 OsSUS1 Os01g69110 -
Sgf73 ATXN7 AtSGF73 ND OsSGF73 ND -
SPT Spt3 SPT3 AtSPT3 At1g02680 OsSPT3 Os01g23630 Seed development [100]
Spt7 STAF65/ SUPT7L AtSPT7 At1g32750 OsSPT7 Os06g43790 -
Spt8 ND AtSPT8 ND OsSPT8 ND -
Spt20 (Ada5) SPT20 AtSPT20 At1g72390 OsSPT20 Os01g02860 Photoperiodic flowering regulation [101, 102]
TAFs Taf5 TAF5L AtTAF5 At5g25150 OsTAF5 Os06g44030 Plant viability; Male gametogenesis; Pollen tube development [90]
Taf6 TAF6L AtTAF6 At1g04950 OsTAF6 Os01g32750 Plant viability; Pollen tube growth [83]
AtTAF6b At1g54360 -
Taf9 TAF9 AtTAF9 At1g54140 OsTAF9 Os03g29470 -
TAF9b OsTAF9b Os07g42150 -
Taf10 TAF10 AtTAF10 At4g31720 OsTAF10 Os09g26180 Salt tolerance during seed germination [103]
Taf12 TAF12 AtTAF12 At3g10070 OsTAF12 Os01g63940 -
AtTAF12b At1g17440 OsTAF12b Os01g62820 Negative response to ethylene and cytokinin signaling [104, 105]
Other Subunits Chd1 ND AtCHR5 At2g13370 OsCHD1 OsJ_25446 Embryo development; Seed maturation [106]
Sgf29 STAF36 AtSGF29a At3g27460 OsSGF29 Os12g19350 Flowering initiation, Auxiliary role in salt stress [61]
AtSGF29b At5g40550 -
Tra1 TRRAP AtTRA1a At2g17930 OsTRA1 Os07g45064 -
AtTRA1b At4g36080 -

ND: Not detected.

Alignment and phylogenetic analysis

Clustal-X version 1.83 software program was used for multiple sequence alignment of the protein sequences [33]. The aligned sequences were further used as input to create phylogenetic trees with the Neighbor-Joining method using a Jones-Taylor-Thornton (JTT) model. Bootstrapping was performed, involving 1000 replicates, to represent the evolutionary history of the group analyzed. The evolutionary distance was computed in MEGA 6.06 version [34].

Domain analysis and chromosomal localization

The domain analysis was performed by CDD (Conserved Domain Database) and Pfam (protein families database) with an e-value 1.0. Chromosome Map Tool database was used to define the position of the SAGA complex genes on Arabidopsis chromosomes [35]. "Paralogous in Arabidopsis" were used for determining the gene duplications and their existence of duplicated segments on chromosome with parameters set to a threshold above 6 per block for paired proteins [36].

Conserved motif analysis

The cis-regulatory elements/motifs were analyzed in 1000 bp upstream from the transcription start site (TSS) by using web based database Plant cis-acting regulatory DNA elements (PLACE) and Plant Cis-Acting Regulatory Elements (PlantCARE) databases and portals [37, 38].

In silico microarray expression and protein interactome analysis

Microarray experiments data from Genevestigator database and analysis toolbox were employed to determine the gene expression profile of Arabidopsis and O. sativa SAGA complex genes in different tissue [39]. The cDNA signatures from Massively Parallel Signature Sequencing (MPSS) were used to count the number of corresponding mRNA molecules produced by each gene of Arabidopsis and O. sativa SAGA complex [40]. A protein-protein interaction network, for the prediction of functional associations within SAGA complex proteins, was prepared using the STRING database with a confidence threshold score of 0.6. [41]. The network was showed in the ‘evidence’ view, whereby lines linking proteins signify the category of evidence used in anticipating the association or interaction.

Functional annotation and co-expression analysis

Functional annotation and Gene Ontology analysis were performed from TAIR and agriGO [35, 42]. Co-expression analysis for gene pairs and co-expressed gene network analysis for each SAGA gene was acquired from ATTED-II (The Arabidopsis trans-factor and cis-element prediction database) version c4.1 [43].

RNA extraction and Real-time PCR analysis

Total RNA was extracted from the flowers, leaves, roots, seedlings, stems and siliques as well as from treated leaves by Sigma’s Spectrum plant total RNA isolation kit. The integrity of RNA, after DNase I treatment, was confirmed by agarose gel electrophoresis. Two microgram of total RNA was used as a template for first-strand cDNA synthesis using the Superscript-II RT kit (Invitrogen). Real-time PCR (qRT-PCR) gene expression analysis was performed and detected by using an ABI’s 7500 Fast Real-time PCR machine [44]. Gene specific forward and reverse primers were designed by using ABI’s-Primer express v2.0 software (S3 Table). The transcripts were normalized using Ubiquitin-10 (Ubq10, At4g05320) transcripts that work as internal control. The relative expression level of target genes was analysed by ΔΔCt method.

Results

Identification and classification of SAGA complex subunits in plants

The SAGA complex is a multiple subunit protein complex and is highly conserved among human, S. cerevisiae and Drosophila [13, 17]. The putative SAGA genes were identified in Arabidopsis and O. sativa genomes using protein sequences of S. cerevisiae and human SAGA genes as queries against the protein databases of Arabidopsis and O. sativa (NCBI, TAIR and RAP) (S1 and S2 Tables).

We identified four protein subunits in the ADA group of the SAGA complex, viz. ADA1, ADA2b, ADA3 and GCN5 (ADA4) (Table 1 and S2 Table). ADA2b (At4g16420) and GCN5 (At3g54610) have been previously studied in plants [7, 28, 30, 45, 46]; however, ADA1 and ADA3 proteins are yet to be characterized in plants. Two ADA1 proteins were identified each in Arabidopsis (At2g14850 and At5g67410) and O. sativa (Os12g39090 and Os03g55450) genome as homologs of S. cerevisiae and human ADA1 (Table 1 and S2 Table). Similarly, Arabidopsis (At4g29790) and O. sativa (Os01g73620) ADA3 were identified as homologs of S. cerevisiae and human ADA3 (Table 1 and S2 Table).

S. cerevisiae and human SAGA complex contain four proteins in DUBm group, which mainly participate in the histone deubiquitylation and mRNA export [47], however, we identified three out of four proteins in Arabidopsis and O. sativa (Table 1). Arabidopsis USP22 (At5g10790) and O. sativa UBP22 (Os04g55360) proteins were found as homologs of S. cerevisiae UBP8 and human USP22, respectively (S2 Table). Arabidopsis (At5g58575) and O. sativa (Os05g28370) SGF11 proteins were identified as homologs of human ATXN7L3 and S. cerevisiae SGF11 (Table 1 and S2 Table). Notably, S. cerevisiae SGF11 and human ATXN7L3 (S. cerevisiae SGF11 homolog) share low (15.3%) similarity between their protein sequences [48]. Arabidopsis (At3g27100) and O. sativa (Os01g69110) SUS1 showed significant homology with the corresponding S. cerevisiae SUS1 and human ENY2.

In S. cerevisiae and human, three to four proteins- SPT3, SPT7, SPT8 (not present in humans) and SPT20, have been reported in the SPT group of the SAGA complex (Table 1). Our study identified SPT3 and SPT20 proteins in Arabidopsis and O. sativa. Interestingly, the human SPT3 displays extensive sequence similarity to the histone fold motifs of TAF13 in its N-terminal region [49, 50]. We found conserved domain TAF13 in Arabidopsis At1g02680 and O. sativa Os01g23630 (Table 1 and S2 Table). The SPT20 domain was found to be conserved in Arabidopsis At1g72390 and O. sativa Os01g02860 proteins (Table 1 and S2 Table). In earlier studies, a low level of similarity was reported between SPT3 (30%) and SPT20 (32.5%) homologs of S. cerevisiae and human (S2 Table) [51, 52]. The SPT7 protein contains Bromo-domain, a motif found in several transcription factors and co-activators, which is responsible for the acetylation of histones and transcriptional activation [5355]. In Arabidopsis, 29 Bromo-domain-containing proteins are reported [56]. The BLAST analysis suggested that Arabidopsis At1g32750 (e-value 3e-07) and O. sativa Os06g43790/Os02g38980 (e-value 2e-07/1e-07 and protein similarity 29 /25%, respectively) have the highest protein sequence similarity to S. cerevisiae SPT7 and particularly to its Bromo-domain region. However, human STAF65/SUPT7L (homolog of yeast Spt7) BLAST analysis revealed extremely low protein similarity and insignificant e-value of the search Spt7 homolog in Arabidopsis and rice genome. SPT8 protein of S. cerevisiae contains WD40 domain repeats and facilitates TBP interaction [8]. Arabidopsis and other plants encompass more than 200 putative WD40 domain containing proteins [57]. Arabidopsis At5g08390 and At5g23430 displayed protein similarity with corresponding S. cerevisiae SPT8. However, in plant genome, a large number of plant proteins comprising either Bromo-domain or WD40 domain, exhibited a substantial level of similarity with the Bromo-domain for SPT7 and the WD40 domain for SPT8, henceforth, further biochemical evidence is required to validate these subunits of the SAGA complex in the two plant species, Arabidopsis and O. sativa.

Interestingly, several TAFs subunits are shared by several complexes like TFIID, SAGA, SLIK (SAGA-like complex), and STAGA (SAGA altered, SPT8 absent) as earlier reported in S. cerevisiae and human [58]. Lago et al., 2004 explained about different TAFs and their conserved domain structures in Arabidopsis [59]. The TAF proteins in the SAGA complex include- TAF5, TAF6, TAF9, TAF10 and TAF12. However, our genome-wide similarity search analysis identified two candidate proteins representing TAF12 in O. sativa (Table 1), unlike only one protein reported previously [59].

Apart from these four groups, some other components also present in the SAGA complex, such as CHD1 (chromo-domain helicase DNA binding protein 1), TRA1 (Transcription-associated protein 1) and SGF29 (SAGA-associated factor 29) (Table 1). The CHD subfamily-I chromatin remodeling proteins, S. cerevisiae CHD1 and human CHD2, share 45% protein similarity (S2 Table) [60]. BLAST searches identified Arabidopsis CHR5 (At2g13370) and O. sativa CHD (OsJ_25446) as homologs of S. cerevisiae CHD1 and human CHD2 (Table 1 and S2 Table). Further, we also identified two proteins, At3g27460 and At5g40550 in Arabidopsis encoding SGF29, as reported recently [61] and one protein in O. sativa (Os12g19350) (Table 1 and S2 Table). TRA1 is a representative of a group of proteins that include DNA-dependent protein kinase catalytic subunit, ATM (Ataxia telangiectasia mutated) and TRRAP (transformation/transcription domain-associated protein), with the carboxyl-terminal regions related to phosphatidylinositol 3-kinases [62]. We identified two TRA1 protein orthologs in Arabidopsis (At2g17930 and At4g36080) and one in O. sativa (Os07g45064) with the corresponding S. cerevisiae TRA1 and human TRRAP (Table 1 and S2 Table). In some reports, RTG2 protein has been considered as a subunit of the SAGA complex [47], whereas sometimes it has been suggested as a variant of the SAGA complex, SLIK [1, 17, 63]. Further biochemical evidences are required to validate the presence of RTG2 in Arabidopsis and O. sativa SAGA/SLIK complex.

Conserved domains in plant SAGA complex

The protein domains of Arabidopsis and O. sativa SAGA subunits, identified with the corresponding domains of S. cerevisiae and human SAGA subunits, is presented in Fig 1 and S1 Fig Intriguingly, numerous known structural features of protein domains in Arabidopsis and O. sativa SAGA complexes are common with S. cerevisiae and human SAGA complexes. These include HAT module, WD repeat domain, histone fold domains, DUBm, interaction and structural integrity protein domains. The protein similarity of each domain of the SAGA complex shows evolutionary conservation across the species (Table 2). The domains of plant SAGA components share moderate (30–50%) to the high (50% and above) similarity with their counterparts in S. cerevisiae and human excluding FAT-domain and chromo-domain (Table 2 and S1 Fig).

Fig 1. Domain organization of representative SAGA complex proteins in Arabidopsis and Oryza sativa.

Fig 1

The positions of conserved domains which are typical for SAGA are shown. Domain abbreviations are: BR: Bromodomain; NAT_SF: N-Acyltransferase superfamily; ZZ_ADA: ZZ-type Zinc finger; SANT: SWI3, ADA2, N-CoR and TFIIIB DNA-binding domains; SAGA-TAD1: Transcriptional regulator of RNA pol II, SAGA, subunit; ADA3: ADA3 superfamily Histone acetyltransferases subunit 3; TRRAP: TRansformation/tRanscription domain-Associated Protein (TRRAP), pseudokinase domain; FAT: FRAP, ATM and TRRAP domain; FATc: FRAP, ATM, TRRAP C-terminal; TAF5-NTD2: N-terminal region of TATA Binding Protein (TBP) Associated Factor 5; WD-40: Trp-Asp (W-D) dipeptide 40 amino acid motifs; TFIID-18: Transcription factor II D 18kDa subunit; PEPTIDASE C19D: Peptidase C19 contains ubiquitinyl hydrolases; Zn-UB: Zn-finger in ubiquitin-hydrolases; ENy2: enhancer of yellow; DEXDc: DEAD-like helicases superfamily; CHROMO: Chromatin organization modifier (chromo) domain; HELICc: Helicase superfamily c-terminal domain; SGF11: SaGa associated Factor 11; SWIRM: SWI3, RSC8 and MOIRA. The number represents the amino acids in domain and protein.

Table 2. Domain similarities in SAGA complex protein among Arabidopsis, human, O. sativa and S. cerevisiae.

SAGA Subunits Domain Arabidopsis thaliana Oryza sativa Human
Human S. cerevisiae O. sativa Human S. cerevisiae S. cerevisiae
ADA1a SAGA-TAD1 33 40 57/60 a 36 37 36
ADA1b SAGA-TAD1 35 39 50/60 a 36 41 -
ADA2b ZZ_ADA2 47 64 89 57 69 51
SANT 69 68 83 64 70 73
SWIRM 63 60 76 58 54 50
ADA3 ADA3 45 46 51 39 44 45
GCN5(ADA4) BROMO 72 63 83 72 59 63
NAT_SF 71 80 89 72 81 74
SPT3 TAF13 57 60 74 50 50 63
SPT20(ADA5) SPT20 48 33 62 42 34 34
TAF5 TAF5_NTD2 62 59 88 62 63 63
WD40 50 51 94 49 51 66
TAF6 TAF6 62 58 88 61 60 57
TAF6b TAF6 57 54 80 - - -
TAF9 TAF9 73 64 73 62 53 60
TAF9b TAF9 - - 68 63 54 -
TAF10 TAF10 69 55 87 66 56 53
TAF12 TAF12 54 53 62/59 a 83 66 69
TAF12b TAF12 76 68 83/85 a 82 64 -
TRA1a FAT 56 57 27 20 17 59
TRRAP 59 58 93 56 57 57
FATc - 55 94 - 52 -
TRA1b FAT 55 48 27 - - -
TRRAP 58 57 91 - - -
FATc - 54 97 - - -
SGF29a SGF29 49 48 82 52 47 36
SGF29b SGF29 48 47 84 - - -
UBP8 PEPTIDASE-C19 60 49 74 60 47 52
SGF11 SGF11 70 76 91 69 73 61
SUS1 ENY2 76 76 89 81 81 100
CHD1/CHR5 CHROMO b 19/35 18/34 72 21/33 14/32 18/16
DEXDc 78 70 91 82 75 70
HELICc 79 87 86 91 77 73

aFor Arabidopsis first protein then second protein domain similarity percentage with O. sativa first protein and second protein domain given.

bFor two domains of Chromo present in S. cerevisiae and human.

Phylogenetic and evolutionary analysis of the SAGA complex family among different organisms

To investigate the evolutionary association among Arabidopsis and O. sativa SAGA complex proteins, phylogenetic trees were made from the alignments of their full-length protein sequences together with SAGA complex proteins of mammals (Homo sapiens, Mus musculus and Rattus norvegicus), an arthropod (Drosophila melanogaster), fungi (S. cerevisiae and Schizosaccharomyces pombe), dicot plants (A. thaliana, A. lyrata, Glycine max, Medicago truncatula, Populus trichocarpa, Ricinus communis, and Vitis vinifera), monocot plants (Brachypodium distachyon, O. sativa, Sorghum bicolor and Zea mays), a tracheophyte (Selaginella lepidophylla), a bryophyte (Physcomitrella patens) and algae (Chlamydomonas reinhardtii and Ostreococcus lucimarinus). In order to evaluate the molecular evolutionary relationship and conservation among SAGA protein components in different organisms, we aligned the different SAGA subunits and constructed a phylogenetic tree for each group. The phylogenetic tree analysis inferred immense conservation among the SAGA protein domains in S. cerevisiae, mammals, Arabidopsis, O. sativa, algae, bryophyte and Drosophila (Figs 2 and 3; Table 3 and S2 Fig). In the case of ADA group, three clades were exhibited for each SAGA subunit. The first and second clades comprised GCN5 (ADA5) and ADA1 proteins, respectively, while the third clade further divided into sub groups- ADA3 and ADA2b (Fig 2). In the phylogenetic analysis of ADA proteins from various organisms fall in a similar clade, excluding SpADA3 and DmADA3, which were close to the ADA1 clade (Fig 2). Similar to ADA group, other groups made several clades based on their similar protein domain specific phylogenetic tree analyses (Fig 3). In the phylogenetic tree of TAFs group, CrTAF5 and OlTAF12 proteins were present in different clades (S2 Fig). The phylogenetic tree constructed from plant SAGA proteins revealed that these proteins diverge into monocots and dicots (Figs 2 and 3). Based on the phylogenetic tree analysis, most protein domains in the SAGA subunits were remained extremely conserved in S. cerevisiae, mammals, Arabidopsis, O. sativa, algae, lycopsida, bryophyte and Drosophila during the course of evolution (Table 3).

Fig 2. Phylogenetic relationship of the ADAs protein of the SAGA complex.

Fig 2

ADAs protein sequences were used from At, Arabidopsis thaliana (red circle); Dm, Drosophila melongaster; Hs, Homo sapiens (red square); Os, Oryza sativa (red diamond shape); Mm, Mus musculus; Rs, Rattus norvegicus; Sc, Saccharomyces cerevisiae (red triangle); Sp, Schizosaccharomyces pombe; Zm, Zea mays; Rc, Ricinus communis; Pt, Populus trichocarpa; Vv, Vitis vinifera; Al, Arabidopsis lyrta; Mt, Medicago truncatula; Bd, Brachypodium distachyon; Sb, Sorgum bicolor; Sm, Selaginella moellendorffii; Pp, Physcomitrella patens; Cr, Chlamydomonas reinhardtii and Ol, Ostreococcus lucimarinus. Phylogeny reconstruction was analysed by neighbour-joining statistical method. Test of phylogeny was analysed by the bootstrap method (1,000 replicates).

Fig 3. Phylogenetic relationship of DUBm, SPTs and other subunits of the SAGA complex.

Fig 3

Different subunits of the SAGA complex protein sequences were used from At, Arabidopsis thaliana (red circle); Dm, Drosophila melongaster; Hs, Homo sapiens (red square); Os, Oryza sativa (red diamond shape); Mm, Mus musculus; Rs, Rattus norvegicus; Sc, Saccharomyces cerevisiae (red triangle); Sp, Schizosaccharomyces pombe; Zm, Zea mays; Rc, Ricinus communis; Pt, Populus trichocarpa; Vv, Vitis vinifera; Al, Arabidopsis lyrta; Mt, Medicago truncatula; Bd, Brachypodium distachyon; Sb, Sorgum bicolor; Sm, Selaginella moellendorffii; Pp, Physcomitrella patens; Cr, Chlamydomonas reinhardtii and Ol, Ostreococcus lucimarinus. Phylogeny reconstruction was analysed by neighbour-joining statistical method. Test of phylogeny was analysed by the bootstrap method (1,000 replicates). (A) DUBm protein; (B) SPTs protein; (C) CHDs protein; (D) SGF29 protein; (E) TRA1 protein.

Table 3. Putative SAGA complex genes in higher and lower plant organisms.

Gene Symbol Dicots Monocots Lycopsida Bryophyte Green alga
Gm a Al a Mt a Pt a Vv a Rc a Bd a Sb a Zm a Sm a Pp a Cr a Ol a
SPT3 NP_001240103 XP_002889412 XP_003591431 XP_002320312 XP_002275358 XP_002515305 XP_003560386 XP_002455591 NP_001148906 XP_002976827 XP_001759999 XP_001692186 XP_001418060
XP_003535970 - - - XP_003632409 - - - - - XP_001758422 - -
SPT20 XP_003529843 XP_002887433 XP_003627348 XP_002304116 XP_002272317 XP_002529195 XP_003573851 ND ND ND XP_001762074 ND ND
XP_003548371 - XP_003611021 XP_002331186 - - XP_003565261 - - - - - -
ADA1 XP_003555984 XP_002883862 XP_003608475 XP_002330802 XP_002279502 XP_002527493 XP_003580724 XP_002442392 NP_001170067 XP_002988060 XP_001769204 ND ND
XP_003550982 XP_002878766 XP_003588660 XP_002332086 XP_002280562 XP_002525253 XP_003579465 XP_002447621 NP_001143099 XP_002981113 - - -
XP_003549203 XP_002872330 - XP_002313901 XP_002263494 XP_002515336 XP_003579250 XP_002447255 NP_001141662 - - - -
XP_003536588 XP_002869325 - XP_002313900 - XP_002512776 XP_003579249 XP_002452025 NP_001136645 - - - -
XP_003526455 XP_002869187 - XP_002304832 - - - XP_002463839 NP_001132220 - - - -
XP_003525861 XP_002865004 - XP_002300259 - - - - - - - - -
XP_003523718 - - XP_002300258 - - - - - - - - -
XP_003545542 - - XP_002297698 - - - - - - - - -
ADA2b XP_003534737 XP_002868135 XP_003594266 XP_002323129 XP_002262737 XP_002522899 XP_003559501 XP_002463870 NP_001105146 XP_002972238 XP_001755499 ND XP_001422948
XP_003547285 - - XP_002307906 XP_002268970 XP_002510307 - - NP_001105664 - XP_001784968 - XP_001420946
XP_003544007 - - XP_002320515 - - - - - - - - -
ADA3 XP_003536708 XP_002869409 ND XP_002311946 XP_002265763 XP_002525000 XP_003572250 XP_002445585 ND XP_002968380 XP_001782560 ND ND
XP_003539168 - - - - - - - - XP_002965949 - - -
XP_003555871 - - - - - - - - - - - -
GCN5 XP_003520580 XP_002876262 XP_003628592 XP_002306812 XP_002275146 XP_002520973 XP_003573924 XP_002464623 NP_001105145 XP_002960878 XP_001766378 XP_001696370 XP_001419344
XP_003553477 - - - - - - - - XP_002967134 - - -
UBP22 XP_003550210 XP_002871441 XP_003588879 XP_002309685 XP_002283376 XP_002515408 XP_003579384 XP_002448634 NP_001132802 XP_002973842 XP_001765324 XP_001692784 XP_001420878
XP_003544592 - XP_003609454 XP_002324922 XP_003633155 XP_002530760 - - - XP_002983550 - XP_001702430 XP_001420231
XP_003549730 - - XP_002324616 - - - - - - - - -
XP_003542653 - - - - - - - - - - - -
SGF11 NP_001241902 XP_002864581 XP_003594873 XP_002299995 XP_003632167 XP_002516646 XP_003568653 XP_002439607 NP_001144045 XP_002963785 XP_001779739 XP_001703794 ND
NP_001241613 - XP_003603196 XP_002313242 - - XP_003575785 - - XP_002974874 XP_001754483 - -
- - - - - - - - - - XP_001760795 - -
ENY2 XP_003547647 XP_002877018 ND XP_002328888 XP_002269535 XP_002509517 XP_003564943 XP_002458995 NP_001148745 XP_002960528 XP_001764723 XP_001701309 ND
NP_001236339 - - XP_002298626 XP_002514922 - - - XP_002967190 XP_001759104 - -
CHR5 XP_003519517 XP_002885872 XP_003617298 XP_002313369 XP_002275100 XP_002531123 XP_003562521 XP_002463329 NP_001105087 XP_002969372 XP_001767461 XP_001703254 XP_001418535
XP_003545390 - XP_003600162 - - - - - - XP_002970703 XP_001782004 - -
TAF5 XP_003526182 XP_002872141 XP_003603301 XP_002309672 XP_003631761 XP_002515435 XP_003563321 ND NP_001183382 XP_002974112 XP_001769775 XP_001696990 XP_001420161
XP_003522395 - - XP_002324907 XP_002285276 - - - - XP_002968859 - - -
XP_003549326 - - - - - - - - - - - -
TAF6 XP_003518649 XP_002892254 XP_003621904 XP_002320500 XP_002264290 XP_002528944 XP_003577929 XP_002459984 ACL54361 XP_002969840 XP_001762306 XP_001692591 XP_001421048
XP_003551737 - XP_003600186 XP_002298845 XP_002276969 XP_002531209 - - - XP_002985176 - - -
XP_003551827 XP_002891885 - - - - - - - - - - -
TAF9 NP_001236385 XP_002894479 XP_003598543 XP_002299587 XP_002273931 XP_002521322 XP_003557734 XP_002467704 NP_001130845 XP_002967605 XP_001785776 XP_001702038 XP_001421943
NP_001235586 XP_002891803 XP_003635996 - - - XP_003578007 - - - - - -
TAF10 NP_001236890 XP_002867282 XP_003624232 XP_002324360 XP_002267115 XP_002515379 XP_003557439 XP_002462422 NP_001148356 XP_002975098 XP_001781637 XP_001697833 XP_001415768
XP_003524747 - - - XP_002266754 - - XP_002460155 - XP_002963932 - - -
TAF12 XP_003542594 XP_002884775 XP_002308150 XP_002304140 XP_002277150 XP_002528715 XP_003564657 XP_002458799 NP_001169752 XP_002975297 XP_001781440 XP_001692926 XP_001415532
XP_003528481 XP_002892951 XP_003627263 XP_002299594 - XP_002521336 XP_003577014 XP_002458756 - - - - -
TRA1 XP_003517177 XP_002867036 XP_003612164 XP_002327756 XP_003631895 XP_002521662 XP_003559884 XP_002463283 NP_001105293.1 XP_002972813 XP_001764071 XP_001701957 XP_001419308
XP_003537633 XP_002886137 - XP_002304328 - - - - - XP_002984389 - - -
SGF29 XP_003547706 XP_002870694 ND XP_002298555 XP_003633806 XP_002521490 XP_003576694 XP_002443131 NP_001141068 XP_002968194 XP_001755688 ND XP_001417743
XP_003547704 XP_002877055 - - XP_003633807 - - - - - XP_001785583 - -
XP_003553408 - - - - - - - - - - - -
XP_003547705 - - - - - - - - - - - -

aGm, Glycine max; Al, Arabidopsis lyrta; Mt, Medicago truncatula; Pt, Populus trichocarpa; Vv, Vitis vinifera; Rc, Ricinus communis; Bd, Brachypodium distachyon; Sb, Sorgum bicolor; Zm, Zea mays; Sm, Selaginella moellendorffii; Pp, Physcomitrella patens; Cr, Chlamydomonas reinhardtii and Ol, Ostreococcus lucimarinus.

ND: Not detected.

The phylogenetic trees were also constructed using the representative domain sequences of each protein of the SAGA complex of Arabidopsis, D. melanogaster, mammals (H. sapiens and M. musculus), O. sativa and S. cerevisiae (S3 Fig). The analysis of phylogenetic tree from different domain of ADA protein groups of the SAGA complex showed two clades, the first clade comprised of ADA3, GCN5, SWRIM-ADA2b and SANT-ADA2b that represents HAT modules of the SAGA complex. However, the ZZ-ADA2b domain, which is involved in interaction with GCN5, presented with ADA1 domains in the second clade (S3A Fig). The phylogenetic tree analysis of full length protein sequences indicated that ADA1 forms a different group from other ADA protein groups (Fig 2). The apparent reason behind the presence of two ZZ-ADA2b and ADA1 domains in one group might be that both protein domains are involved in protein-protein interactions. The phylogenetic tree constructed from each domain of the DUBm SAGA complex subunits suggested that SGF11 and Peptidase C19D-UBP domains were present in the same clade (S3C Fig), which is against the result obtained in the phylogenetic tree with full length protein (Fig 3A). TAFs domain grouped according to their domain features, for example, histone-fold domain containing TAF6, TAF9 and TAF12 domain were present in the same clade, in which TAF6 and TAF9 contains similar histone-fold domain (S3G Fig) [59]. TAF5 and TAF10 proteins were present in the same sub-group (S2 Fig) and their domains (NTD-TAF5 and TAF10) also showed a close relation (S3G Fig).

Notably, several paralogs were found for SAGA complex components in selected plants, mainly in G. max and P. trichocarpa (Table 3). Moreover, a variation was observed in the total number of SAGA complex components among dicots, as compared to monocots (Table 3). The variation in the number of the SAGA complex subunits suggested that these components could have been executed to accomplish the distinct and specialized roles in plants.

Chromosomal distribution and functional annotation of plant SAGA complex

The Arabidopsis Genome Initiative provides the opportunity to identify the instances of chromosomal block duplication in the genome [64]. We intended to investigate, whether proteins encoding for the SAGA complex are associated with chromosomal block duplication in Arabidopsis. We used TAIR chromosome map viewer and Paralogons in Arabidopsis for the localization of the SAGA components across the five chromosomes (S4 Fig). Most of these SAGA complex proteins were in the duplicated segmental regions of Arabidopsis chromosome [36]. Moreover, we also identified that some of the Arabidopsis and O. sativa SAGA subunits were found in more than one copy such as ADA1, TAF6, TAF9, SFG29 and TRA1. Thus, it seems that some of the SAGA proteins were duplicated during evolution.

The functional characterization analysis showed that Arabidopsis and O. sativa SAGA complex components play a key role in gene expression, transcription initiation, complex assembly and several metabolic and cellular processes (Fig 4). Gene Ontology predicted that plant SAGA complex components also participate in a transcription regulator activity, binding, catalytic activity as well as in the development of cell and organelle parts (S4 Table). Recent studies suggest participation of some of the plant SAGA complex subunits, for example Arabidopsis ADA2B, SGF29 and GCN5, in the light- [29], cold- [28, 65] and salt-induced [61] gene expression, flower development [66], histone acetylation [30, 45]. The functional characterization analysis also indicated that their involvement in auxin, cytokinin, ethylene and jasmonic acid mediated signaling pathways (S4 Table). In sum, functional and GO analysis predicated the involvement of the plant SAGA complex not only in chromatin remodeling, but also in abiotic and biotic processes.

Fig 4. Functional annotation of plant SAGA complex components.

Fig 4

(A) Arabidopsis (B) O. sativa. Functional annotations were performed by TAIR and agriGO databases. The percentage (%) associated with each annotation indicates the percentage of segments annotated to that category.

Protein-protein interactome analysis of Arabidopsis SAGA complex

To examine interactions among Arabidopsis SAGA complex components, we mapped the SAGA proteins over STRING interactome, a database of known and predicted protein interactions [41]. The analysis of Arabidopsis SAGA component proteins revealed an interconnected sub-network of 131-hub proteins (confidence score 0.6, Fig 5 and S5 Table). These analyses suggested that many hub proteins create a network which behaves as a functional module within the complex. Moreover, the protein-protein interaction analysis of S. cerevisiae SAGA proteins using the STRING database displayed 190-protein interactions with a confidence score of 0.6 (S6 Table). Interestingly, most of these protein-protein interactions were similar in the SAGA proteins of Arabidopsis and S. cerevisiae (S5 and S6 Tables). The mutation and biochemical characterization studies in S. cerevisiae and mammals established that these interactions are essential for SAGA structure and its stability. For instance, any alteration in SPT7, SPT20, TAF5, TAF10, or TAF12 affects the SAGA composition and integrity [6769].

Fig 5. Interactome of the SAGA complex subunits.

Fig 5

SAGA complex subunits interactomes were obtained from STRING database. Interactome between the protein pair is shown.in a confidence view where associations are represented by blue and black lines. Blue lines suggested the binding and black lines suggest a reaction between the proteins. The protein-protein network among SAGA component genes was analysed with high confidence of score 0.6.

In silico expression analysis of the SAGA complex encoding genes

Gene expression profiles of the SAGA complex components can provide significant evidences for their potential functional roles. The functional annotation of SAGA components in Arabidopsis and O. sativa revealed their diverse roles in plant development (Fig 4). To further validate, we examined the expression profile of the SAGA complex components in different tissues using Genevestigator microarray database and its expression meta-analysis tool [54], and MPSS database [55]. The expression profile of the SAGA complex encoding genes was examined in 9 different plant organs of Arabidopsis and O. sativa (Fig 6). AtTaf10, AtGcn5 and AtChr5 were expressed at low levels in all the examined developmental stages (Fig 6A). However, the expression of AtAda1a, AtTaf12b, AtTaf6b and AtTra1a was higher in the aforesaid developmental stages. In the case of O. sativa, SAGA subunit genes were found highly expressed in booting, seedling, milk, flower and stem elongation stages (Fig 6B). During germination, transcript accumulation was observed at higher levels for AtTaf6/6b, AtTra1a, AtAda2b, AtTaf1a, AtTaf12b and AtUbp22 genes in Arabidopsis, whereas, for OsAda2b, OsAda3, OsTaf5, OsTaf12/12b, OsTaf1a, OsUbp22, OsSus1, OsSgf11 and OsTaf9 genes in O. sativa. In the booting stage of O. sativa, OsTaf5, OsTaf13, OsTaf12/12b, OsGcn5, OsAda2b, OsAda1a, OsSgf29, OsAda3, and OsSgf11 were among the highly expressed genes, whereas, OsSus1, OsSgf29, OsUbp22, OsTaf6 and OsSgf11 were the genes that highly expressed during dough developmental stages. The meta-analysis displayed an enhanced expression of SAGA component genes in the endosperm (micropylar, peripheral and chalazal), seed coat, suspensor callus and primary cells of Arabidopsis (Fig 7A), whereas in callus, sperm cells, panicle, leaf, pistil, stigma, ovary and root tip of O. sativa (Fig 7B). The results suggest a diverse role of SAGA component genes being expressed throughout different developmental phases in distinct plant organs and tissues.

Fig 6. In silico expression patterns of the SAGA complex genes in developmental tissues.

Fig 6

The expression patterns in different developmental tissues for SAGA genes were obtained from the Genevestigator microarray database tool. (A) Arabidopsis developmental tissues; (B) O. sativa developmental tissues.

Fig 7. In silico expression patterns of the SAGA complex genes in anatomical tissues.

Fig 7

The expression patterns in different anatomical tissues for SAGA genes were obtained from the Genevestigator microarray database tool. (A) Arabidopsis anatomical tissues; (B) O. sativa anatomical tissues.

Data was extracted from the MPSS database library (17 and 20 bases), representing 12 and 13 different anatomical parts of Arabidopsis and O. sativa, respectively. These signatures uniquely recognize specific gene, which show a perfect match (100% identity over the tag length), and signify a quantitative estimation of expression of that gene. These MPSS tags further confirmed transcript abundance of SAGA protein encoding genes in different plant parts (S7 and S8 Tables). Transcript differences are generally presented by the total number of tags (TPM, transcripts per million), low expression if smaller than 25 TPM, moderate expression if 26 to 250 TPM, while highly expressed in case of >250 TPM. Based on these signatures/tags, five Arabidopsis genes viz., AtAda3, AtAda1b, AtTaf12, AtTra1a and AtSgf29 were expressed at low levels, whereas AtTaf10 expressed at a higher level in leaf, root, siliques and callus (S7 Table). Other Arabidopsis SAGA genes exhibited a moderate level of transcript accumulation. MPSS analysis in O. sativa showed that OsAda2b, OsTaf10 and OsTra1 expressed at higher levels (>250 TPM). The maximum transcript abundance was observed for OsAda2b in mature leaves and for OsTaf10 in young leaves, ovary and mature stigma and callus, whereas OsTra1 was significantly expressed in most of the plant parts, except germinating seed and stem. The SAGA genes, OsGcn5, OsAda1a, OsSpt20, OsSgf11, OsTaf9b and OsTaf12 expressed at low levels, whereas others at moderate levels (S8 Table).

Co-expression analysis for gene pairs and gene network analysis of the SAGA complex

The expression profiles of SAGA components in Arabidopsis and O. sativa using Genevestigator and MPSS revealed that many of the components have distinct tissue-specific expressions. We further examined whether these genes co-express during plant development or in any other physiological condition. The co-expression data for each SAGA component gene pairs were generated from ATTED-II database, which includes 1388 microarray experimental data [43]. The strength of co-expression for the interconnecting gene pairs was determined by Mutual Rank (MR) process using these microarray data. Forty-two significant co-expression patterns (Table 4) were obtained between SAGA components from 171 co-expressing gene pairs (S9 Table). These co-expression patterns were identified under different biotic, abiotic, hormonal and tissue conditions, for example, co-expression analysis of gene pairs data showed that Ada2b was strongly co-expressed with Taf6, Taf13 and Spt20 genes in all the developmental and environmental stress conditions. Likewise, Ubp22 co-expressed with Sgf29b and Taf1b with Tra6 and Taf13, at high MR values. The significant MR values for Taf1b, Spt20, Tra1, Taf9, Gcn5, Ada2b and Ada3 suggest their co-expression at the tissue level. The genes, Spt20, Ada2b, Taf12b, Chr5, Taf9 and Taf10 showed co-expression in abiotic stress conditions (Table 4). Under hormonal condition, Ada2b, Ada3, Chr5, Taf6, Taf10, Taf12b, Taf13 and Tra1a exhibited a substantial level of co-expression strength, whereas Spt20 was found to be co-expressed with Chr5 in biotic stress condition (Table 4).

Table 4. Co-expression analysis of SAGA component genes in Arabidopsis.

S.N. LOCUS1 LOCUS2 Mutual Rank (MR) a
All Tissue Abiotic Biotic Hormone
1 At1g04950 TAF6 At4g16420 ADA2B 42.5 706.1 469.5 2967.2 59.4
2 At1g02680 TAF13 At4g16420 ADA2B 47 143.4 451.7 706.6 10.2
3 At1g72390 SPT20 At4g16420 ADA2B 59 625.7 59.7 532.6 555.5
4 At5g10790 UBP22 At5g40550 SGF29B 100 4698.1 223.4 3121.7 8550.2
5 At1g04950 TAF6 At2g17930 TRA1A 137.2 150.1 1762 849.3 3776.9
6 At1g02680 TAF13 At2g17930 TRA1A 158.3 24.1 6755.4 1645.4 6751.4
7 At1g72390 SPT20 At4g29790 ADA3 164.7 121.5 720.7 2500.2 1786.6
8 At1g32750 TAF1B At4g16420 ADA2B 187 554.3 613.6 2615.8 10053.6
9 At2g13370 CHR5 At4g16420 ADA2B 187.3 1798.5 270.9 358.2 2263.7
10 At1g54140 TAF9 At3g54610 GCN5 256.6 92.3 5502.3 3446.5 2020.6
11 At1g32750 TAF1B At1g72390 SPT20 258.8 5.5 1702.5 1398.6 17199.5
12 At1g17440 TAF12B At2g13370 CHR5 261.8 3768.4 129 1889.8 19.6
13 At1g02680 TAF13 At1g32750 TAF1B 285.5 744.5 2116 2013.9 1885.3
14 At1g72390 SPT20 At2g13370 CHR5 296.7 959.4 206.5 140.8 3661.9
15 At4g16420 ADA2B At4g29790 ADA3 315.6 2734.4 966.2 976.3 84.4
16 At1g02680 TAF13 At1g04950 TAF6 330.5 346.1 1827.7 1061.4 523.2
17 At1g02680 TAF13 At3g27100 SUS1 334.8 2570.5 345.5 508.5 6808.6
18 At1g32750 TAF1B At2g17930 TRA1A 339.7 912.1 889.7 2158.4 5755.3
19 At2g17930 TRA1A At4g16420 ADA2B 358.8 124.2 4677.5 2905.3 1184.4
20 At1g04950 TAF6 At4g31720 TAF10 377.1 864.8 2178.7 634.8 32.6
21 At1g02680 TAF13 At1g54140 TAF9 430.9 142.8 1530.5 15622.6 3374
22 At1g17440 TAF12B At1g72390 SPT20 435.2 1511 1180.6 1791.3 1163.2
23 At2g13370 CHR5 At4g29790 ADA3 469.1 373.4 1177.1 2125 843.3
24 At1g02680 TAF13 At4g31720 TAF10 482 2554 228.8 427.2 1064.2
25 At1g32750 TAF1B At2g13370 CHR5 489.7 1185 3523.9 1579 2548.7
26 At1g72390 SPT20 At2g17930 TRA1A 491.9 929.8 7664.1 2540.6 382.9
27 At1g17440 TAF12B At2g14850 ADA1A 509.9 563.8 2349.7 328 12012.7
28 At1g54140 TAF9 At4g31720 TAF10 543.1 547.6 137.8 18320.2 280.8
29 At1g32750 TAF1B At5g58575 SGF11 573.2 1972.6 1479.7 1786.7 12185
30 At3g10070 TAF12 At5g40550 SGF29B 576.3 1814.2 466.1 1005.4 19138.8
31 At1g17440 TAF12B At4g29790 ADA3 601.2 1368.2 1636.9 2773 572.6
32 At1g54360 TAF6B4 At2g17930 TRA1A 613.1 2589.2 1343.7 1096.5 1074.8
33 At1g72390 SPT20 At2g14850 ADA1A 659.8 224.1 2409.6 2249.2 4698.8
34 At1g17440 TAF12B At2g17930 TRA1A 718.4 1106.6 5512.9 1279.2 117.6
35 At1g02680 TAF13 At2g14850 ADA1A 777.9 446.8 5712.8 6936.2 11359.6
36 At1g32750 TAF1B At2g14850 ADA1A 780.8 129.4 7427.5 1777.1 1345.7
37 At1g17440 TAF12B At4g16420 ADA2B 785.6 1057.4 3349.8 2005.7 1624.9
38 At3g27100 SUS1 At4g31720 TAF10 793.1 3246.7 821 350.5 5879.1
39 At2g14850 ADA1A At4g16420 ADA2B 872.4 652.8 965.4 326.1 12755.7
40 At1g54360 TAF6B4 At3g54610 GCN5 897 1218.2 422.4 1624.3 8621.1
41 At1g32750 TAF1B At4g29790 ADA3 897.7 186.4 9527.1 1397.2 12528
42 At1g02680 TAF13 At1g72390 SPT20 923.8 1201.9 4235.8 744.2 7934.4

a MR values represent here <1000 shows significant co-expression data (in bold) [43]

The co-expressed gene network analysis was done to identify the genes, which co-regulate with the SAGA complex (S5 Fig). Co-expressed gene network provides the evidence of highly interconnected expression modules of a subset of genes, which additionally show another layer of regulation, and consequently the complementary evidences to understand gene function network. A total of 181 proteins was found to be co-regulated with SAGA complex components (S10 Table). Approximately 36% of 181 proteins are recognized to be involved with regards to abiotic or biotic stimulus or stress, developmental processes, transcription regulation, signal transduction and other biological processes (S6 Fig). This analysis further indicates a potential role of the SAGA complex in regulating plant development and responses to various physiological stresses.

Expression analysis of the SAGA complex subunit during developmental stages and stress conditions

We performed a quantitative gene expression analysis of ten representative SAGA components by QRT-PCR in different Arabidopsis tissues: flowers, mature leaves, siliques, six-day-old seedlings, stems and roots (Fig 8A). The gene expression profile of the SAGA complex components was plotted with reference to the expression of ubiquitin. The genes of the SAGA complex, although expressing at a lower level compared with Ubiquitin, showed consistent expression in almost all the examined plant parts (Fig 8A). These indicate the involvement of the SAGA complex in gene regulation throughout the plant body. However, there were certain components that showed spatial preference, for example, the expression of Spt20 was relatively higher in root and leaf, whereas, Sgf11 in leaves and seedlings than other examined tissues.

Fig 8. Real time PCR for Arabidopsis SAGA complex subunits in different development tissues and stress conditions.

Fig 8

(A) The expression pattern of the SAGA complex subunits in different tissues. Relative expression analysis of each was plotted with reference to the expression of ubiquitin (either 6-day-old light-grown seedlings 16 / 8hr, mature leaves, stems, flowers, siliques and roots). (B) The expression pattern of Arabidopsis SAGA complex subunits in response to abiotic salt stress conditions. Arabidopsis Col-0 leaves treated with MS liquid medium (as a control) and NaCl (150 mM in MS media). (C) The expression pattern of Arabidopsis SAGA complex subunits in response to high temperature conditions. Arabidopsis Col-0 leaves kept in MS liquid medium at 22°C (as a control) and 37°C (2 hr in MS media). The asterisks (*) denote P ≤ 0.01.

The effect of high temperature and salt concentrations was also examined on the expression pattern of the SAGA components in Arabidopsis. The excised leaves of Arabidopsis were either exposed to a high temperature at 37°C for 2 hr or 150 mM NaCl for a period of 24 hr for high salt stress condition, and the gene expression of the SAGA components was compared with their respective controls. The gene expression of the most of selected components of the SAGA complex was induced under elevated salt concentration (Fig 8B) and high temperature (Fig 8C); however, the fold of induction varies for different components. Interestingly, Sgf29b expression was suppressed in salt treatment condition (Fig 8B). Thus, the qRT-PCR results suggested the significance of SAGA components gene expression in plants during abiotic stresses.

As discussed above, in silico co-expression analysis of the SAGA complex subunits suggested that these subunits were co-expressed in the tissues, hormones and stress conditions. Notably, the quantitative gene expression analysis of selected SAGA components further supported the co-expression analysis, such as Spt20 and Chr5, Taf13 and Tra1, Spt20 and Chr5 showed high co-expression with significant MR value in tissue; while Spt20 and Ubp22, Sgf11 and Tra1, Spt20 and Chr5, Gcn5 and Taf6 were co-expressed and considerable MR value in abiotic stress. The qRT-PCR analysis is in agreement with the in silico co-expression profile (Table 4, Fig 8 and S9 Table).

SAGA complex regulates expression of heat, salt and light-induced genes

SAGA complex facilitates the PIC assembly in the core promoter region of yeast and human genes [7074]. Little is known about how SAGA complex facilitates gene regulation in plants. To address this, RNA was isolated from seven homozygous T-DNA SAGA subunit Arabidopsis mutants and wild type plants, grown under different conditions such as light/dark, high salt or heat stress (Fig 9 and S7 Fig). The gene expression of light induced (At1g67090 and At4g02770) [2, 75], salt induced (At2g40140 and At1g56600) [76, 77] and heat induced (At1g71000 and At5g12030) [78] genes was examined in these mutant in comparison to the wild type plants by qRT-PCR (Fig 9A). The expression of both the light activated genes was considerably reduced in all the mutants, except in sgf11⁻ for both the genes and in gcn5⁻ for At4g02770, in which the relative expression values were not statistically significant (Fig 9B). In the case of salt stress, the expression level of both the salt induced genes declined in mutants as compared to the wild type, except At1g56600 in taf13⁻, which was statistically not significant (Fig 9C). Under heat stress, expression of the heat activated genes was decreased in mutants, except At5g12030 in gcn5⁻ and taf13⁻ and At1g71000 in sgf11⁻ mutant which were not statistically significant (Fig 9D). These results anticipated that SAGA complex plays significant roles in the transcription regulation of stress inducible genes.

Fig 9. The effect of the SAGA complex on light and stress induced gene expression.

Fig 9

For light condition, RNA was isolated from seedling grown for 5 days and then transfer into light for 4 hr. For salt condition, RNA was isolated from leaves kept in either in 0.5 x MS media (as a control) or in 0.5 x MS media with 150mM NaCl solution for 24 hr. For heat condition, RNA was isolated from Arabidopsis Col-0 leaves kept in MS liquid medium at 22°C (as a control) and 37°C (2 hr in MS medium). Ubiquitin used as internal control. The mark symbols denote (*)—P ≤ 0.001; (#)—P ≤ 0.01. (A) Real time PCR for different selected induced responsive genes in light, salt and heat conditions. (B) The effect of Arabidopsis SAGA complex subunit mutants on light activated gene expression. (C) The effect of Arabidopsis SAGA complex mutant subunits on salt-induced gene expression. (D) The effect of Arabidopsis SAGA complex subunits on heat-induced gene expression.

Discussion

The SAGA complex has been previously shown to be associated with transcriptional regulation of ~10% RNA polymerase II-dependent S. cerevisiae genes, which contribute in response to DNA damage and other stress conditions such as heat, oxidation, and metabolic starvation [71, 72, 79]. A recent report indicates that SAGA complex regulates all active genes and present at their promoters and transcribed regions [80]. With the computational approach, we identified 18 putative SAGA complex subunits in Arabidopsis and O. sativa. The protein similarities among Arabidopsis and S. cerevisiae SAGA complex subunits are low (17%) to medium (51%), as observed between S. cerevisiae and human SAGA complex (15% to 56%; S2 Table). Since the SAGA complex is involved in the fine-tuning of gene expression, this could be one of the reasons for the poor protein similarities. Our results on in silico expression, GO analysis and qRT-PCR of plant SAGA complex representative genes suggested their role in various cellular, physiological and molecular processes. The previous reports on the functions of ADA2b, GCN5, TAF10, TAF6 and SGF29 in plants are in accordance with our study, suggesting conservation of the SAGA complex throughout evolution [28, 46, 61, 8183]. Thus, the presence of conserved domain is helpful in identifying most of the putative members of plant SAGA complexes in different plant organism databases. Beside the low level similarity in full protein sequence (S2 Table), most of the domains present in plant SAGA complex encoding genes were found conserved among different organisms (Figs 2 and 3; Table 2). The similarity between conserved domain’s amino acid sequences of Arabidopsis SAGA was observed higher, i.e. from 30% to 97% (Table 2 and S1 Fig). Notably, similar range of similarities was found between the key domains of the SAGA complex in S. cerevisiae and human (Table 2 and S1 Fig). On the basis of protein or conserved domain similarity and phylogenetic analysis, our results altogether suggested that plant SAGA complex was observed to be closer to the human than that to the yeast SAGA complex (Figs 2 and 3; Table 2 and S2 Table).

Our analysis of protein alignment, phylogenetic tree and chromosomal distribution suggested that many plant SAGA complex representative genes might have duplicated during evolution (Figs 2 and 3; S2 Fig). For example, Taf6, Taf9, Taf12, Ada1, Tra1 and Sgf29 have been found duplicated in either O. sativa or Arabidopsis. Besides these genes, other SAGA subunit genes are also found duplicated in other lower and higher plant groups (Table 3). This duplication event may also lead to variability in the SAGA complex components in plants like Ada2a-containing (ATAC), SLIK/SALSA or STAGA [74], or sometimes shares subunits with other complexes like TFIID [68]. The protein interactome analysis suggested that Arabidopsis SAGA complex proteins interact with each other and thus further suggested their conservation in plants (Fig 5). The structural integrity of the SAGA complex is dependent on the protein-protein interactions as evident in our study, and also discussed in previous reports; such as TAF10 and TAF12 associate directly via their histone fold domains with SPT7 and ADA1, establishing SPT7-TAF10 and ADA1-TAF12 heterodimer, respectively [84, 85], whereas TAF5 interact with ADA1, ADA3 and SPT7 [69].

Our results suggested that the SAGA complex encoding genes expressed in most of the plant parts and playing an essential role in plant development. Previous reports in Arabidopsis, gcn5⁻ exhibit pleiotropic developmental abnormalities, such as abnormal meristem role, dwarfism, loss of apical dominance, defects in floral organ identity [28, 29, 31, 8688]. An insertion of T-DNA elements in the Arabidopsis Ada2b produces a dwarf phenotype with defects in root and shoot development [28, 87, 89]. Arabidopsis sgf29a⁻ shows a little delay in leaf and flower development [61]. Importantly, some reports on plant TAFs (TAF5, 6 and 10) indicated their indispensable role in plant development [81, 83, 90]. Notably, SAGA complex is also critically involved in developmental aspects and is indispensable for viability in metazoan [11]. Recently, ubiquitin protease activity of the SAGA complex showed significant regulation of the expression of the tissues specific genes and developmental processes in Drosophila [91]. In Drosophila, loss of SAGA subunit functions, such as ADA2b, SGF11 and Nonstop protein (homolog of ENY2), display photoreceptor axon targeting defects, whereas, GCN5 has an essential role in the development of eye and wing disc [92, 93]. While, mice TAF9b and GCN5 are required for the regulation of genes during neuronal and mesoderm development [94, 95]. These accumulating evidences indicate that the functions of the SAGA complex in higher organisms involve more sophisticated mechanisms in regulation of gene expression than unicellular counterpart like S. cerevisiae during development processes.

SAGA complex expedites the gene expression that anticipates to various environmental cues such as DNA damage and abiotic stress conditions [12]. Many reports, as discussed above, reveal that the SAGA complex is directly or indirectly contributing in various developmental and stress regulated processes, for example, arsenite stress conditions [52] osmotic stress [96] and ultraviolet induced [97]. The yeast SAGA complex also takes part in the up-regulation of several genes during environmental stress, including carbon starved condition [71]. Our results support the stress inducible expression of several SAGA components in Arabidopsis. Interestingly, the promoter sequence analysis of the SAGA components revealed several stress responsive cis-motifs (S11 Table), indicating their involvement in transcription regulation activities in response to stress. Nevertheless, further experimentation is needed to validate the involvement of these motifs in the regulation of the SAGA component genes. The expression analysis of the SAGA subunits supports its potential roles in response to environmental cues (Figs 6 and 7). These results are in accordance with the earlier published reports on plant ADA2b, GCN5 and SGF29a [26, 28, 46, 61]. Arabidopsis ada2b-1⁻ mutant displays enhanced hypersensitivity to salt and abscisic acid stress than wild-type plants [26, 61]. Although, loss of SGF29a function displays salt stress tolerance, the gene expression level of stress-related genes markers such as COR78 (cold regulated 78), RAB18 (responsive to aba 18), and RD29b (responsive to desiccation 29b) are lower in sgf29a⁻ mutant after 3 hr of NaCl treatment [61]. Arabidopsis HAT protein GCN5 and co-activator ADA2b proteins play significant roles in cold responses and loss of functions of these proteins showed a decline in the expression of several cold-regulated genes [27, 28, 98]. Altogether, the property of the SAGA complex in the regulation of stress genes is not only well maintained within plants, but also comparable to S. cerevisiae or human [71, 74].

In conclusion, we identified 18 subunits of the SAGA complex in Arabidopsis and O. sativa. The protein similarities at the level of conserved domain indicate that the SAGA complex is conserved in eukaryotes such as S. cerevisiae, plants and mammals. The expression analysis of the SAGA components indicates that the networks of SAGA complex are involved in various biological processes in plants, including development, physiology and response to environmental stresses via gene regulation. This study advances our understanding about SAGA components and their different functions in plants.

Supporting Information

S1 Fig. Domain similarity between human, S. cerevisiae, Arabidopsis and O. sativa SAGA complex encoding genes.

Sequence alignments of the representative domains of each protein of the SAGA complex were done by using Clustal X.

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S2 Fig. The phylogenetic relationship of the TAFs group of the SAGA complex.

SAGA complex subunit protein sequences were used from At, A. thaliana (red circle); Dm, D. melongaster; Hs, H. sapiens (red square); Os, O. sativa (red diamond shape); Mm, M. musculus; Rn, R. norvegicus; Sc, S. cerevisiae (red triangle); Sp, S. pombe; Zm, Z. mays; Rc, R. communis; Pt, P. trichocarpa; Vv, V. vinifera; Al, A. lyrta; Mt, M. truncatula; Bd, B. distachyon; Sb, S. bicolor; Sm, S. moellendorffii; Pp, P. patens; Cr, C. reinhardtii and Ol, O. lucimarinus. Phylogeny reconstruction was analyzed by neighbour-joining statistical method based on the JTT matrix-based model. Test of phylogeny was analyzed by the bootstrap method (1,000 replicates). Evolutionary analyses were conducted in MEGA 6.06.

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S3 Fig. Molecular phylogenetic analysis of domains of the SAGA complex components.

Amino acid sequences of domains of the SAGA complex subunits were used from At, A. thaliana; Dm, D. melongaster; Hs, H. sapiens; Os, O. sativa; Mm, M. musculus; Sc, S. cerevisiae. (A) Protein domain of ADAs group; (B) Protein domain of SPTs group; (C) Protein domain of DUBm group; (D) Protein domain of CHD subunit; (E) Protein domain of SGF29 subunit; (F) Protein domain of TRA1 subunit; (G) Protein domain of TAFs group.

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S4 Fig. Chromosomal distribution of the SAGA subunit genes in the Arabidopsis genome.

SAGA encoding genes are plotted on the five Arabidopsis chromosomes according to their sequence spots. The chromosome number is shown at the top of each chromosome and the centromeric regions by constriction on chromosome line bar. Each identical duplicated chromosomal segment is marked by same line colour. The scale is in mega bases (Mb).

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S5 Fig. Co-expressed gene network analysis for Arabidopsis SAGA complex.

The co-expressed gene networks are drawn based on their rank of correlation from ATTED-II database. Orange line displays conserved co-expressed which is inferred from the comparison with mammalian co-expression data provided from COXPRESdb; Red dotted line display protein-protein interaction information that is provided from TAIR and IntAct. The octagon shape indicates transcription factor genes. White circles shape indicates SAGA complex genes which were used to give input for generating gene network. Gray circle shape indicates other genes in co-expressed gene networks.

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S6 Fig. Analysis of the biological process of co-expressed gene network.

A biological process is analyzed by TAIR database using 181 co-expressed genes obtained from ATTED-II database.

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S7 Fig. Characterization of Arabidopsis mutant lines.

Characterization of Arabidopsis chr5‾, gcn5 ‾, sgf11‾, sgf29b‾, taf12b‾, taf13‾ and tra1a‾ T-DNA insertion homozygous mutants were done by qRT-PCR. RNA was isolated from homozygous T-DNA insertion mutants and Col-0 leaves or seedlings.

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S1 Table. Query sequences from S. cerevisiae and human used to search Arabidopsis and O. sativa genome for SAGA gene families.

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S2 Table. Protein similarities of SAGA encoding gene in Arabidopsis, O. sativa, human and S. cerevisiae.

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S3 Table. List of primer used in qRT-PCR.

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S4 Table. Gene Ontology list of Arabidopsis and O. sativa for SAGA complex gene.

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S5 Table. List of Arabidopsis SAGA complex subunits known and predicted protein interactions from STRING database.

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S6 Table. List of S. cerevisiae SAGA complex subunits known and predicted protein interactions from STRING database.

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S7 Table. MPSS data for Arabidopsis SAGA complex encoding genes showing different tissue-specific abundance.

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S8 Table. MPSS data for O. sativa SAGA complex encoding genes showing different tissue-specific abundance.

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S9 Table. List of co-expression genes in Arabidopsis.

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S10 Table. List of genes obtained from ATTED-II for Arabidopsis SAGA complex co-expressed gene network analysis.

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S11 Table. Analysis of cis-regulatory element in 1000bp upstream promoter sequences from TSS in SAGA complex subunit genes using PlantCARE and PLACE database.

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Acknowledgments

The authors are thankful to the CSIR, India for funding under the Institutional and CSIR-EMPOWER (OLP79) project. Rakesh Srivastava is obliged to the CSIR for fellowship support.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by grants from the Council of Scientific and Industrial Research (CSIR), Government of India, under the institutional CSIR-EMPOWER (OLP79) project.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Domain similarity between human, S. cerevisiae, Arabidopsis and O. sativa SAGA complex encoding genes.

Sequence alignments of the representative domains of each protein of the SAGA complex were done by using Clustal X.

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S2 Fig. The phylogenetic relationship of the TAFs group of the SAGA complex.

SAGA complex subunit protein sequences were used from At, A. thaliana (red circle); Dm, D. melongaster; Hs, H. sapiens (red square); Os, O. sativa (red diamond shape); Mm, M. musculus; Rn, R. norvegicus; Sc, S. cerevisiae (red triangle); Sp, S. pombe; Zm, Z. mays; Rc, R. communis; Pt, P. trichocarpa; Vv, V. vinifera; Al, A. lyrta; Mt, M. truncatula; Bd, B. distachyon; Sb, S. bicolor; Sm, S. moellendorffii; Pp, P. patens; Cr, C. reinhardtii and Ol, O. lucimarinus. Phylogeny reconstruction was analyzed by neighbour-joining statistical method based on the JTT matrix-based model. Test of phylogeny was analyzed by the bootstrap method (1,000 replicates). Evolutionary analyses were conducted in MEGA 6.06.

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S3 Fig. Molecular phylogenetic analysis of domains of the SAGA complex components.

Amino acid sequences of domains of the SAGA complex subunits were used from At, A. thaliana; Dm, D. melongaster; Hs, H. sapiens; Os, O. sativa; Mm, M. musculus; Sc, S. cerevisiae. (A) Protein domain of ADAs group; (B) Protein domain of SPTs group; (C) Protein domain of DUBm group; (D) Protein domain of CHD subunit; (E) Protein domain of SGF29 subunit; (F) Protein domain of TRA1 subunit; (G) Protein domain of TAFs group.

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S4 Fig. Chromosomal distribution of the SAGA subunit genes in the Arabidopsis genome.

SAGA encoding genes are plotted on the five Arabidopsis chromosomes according to their sequence spots. The chromosome number is shown at the top of each chromosome and the centromeric regions by constriction on chromosome line bar. Each identical duplicated chromosomal segment is marked by same line colour. The scale is in mega bases (Mb).

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S5 Fig. Co-expressed gene network analysis for Arabidopsis SAGA complex.

The co-expressed gene networks are drawn based on their rank of correlation from ATTED-II database. Orange line displays conserved co-expressed which is inferred from the comparison with mammalian co-expression data provided from COXPRESdb; Red dotted line display protein-protein interaction information that is provided from TAIR and IntAct. The octagon shape indicates transcription factor genes. White circles shape indicates SAGA complex genes which were used to give input for generating gene network. Gray circle shape indicates other genes in co-expressed gene networks.

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S6 Fig. Analysis of the biological process of co-expressed gene network.

A biological process is analyzed by TAIR database using 181 co-expressed genes obtained from ATTED-II database.

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S7 Fig. Characterization of Arabidopsis mutant lines.

Characterization of Arabidopsis chr5‾, gcn5 ‾, sgf11‾, sgf29b‾, taf12b‾, taf13‾ and tra1a‾ T-DNA insertion homozygous mutants were done by qRT-PCR. RNA was isolated from homozygous T-DNA insertion mutants and Col-0 leaves or seedlings.

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S1 Table. Query sequences from S. cerevisiae and human used to search Arabidopsis and O. sativa genome for SAGA gene families.

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S2 Table. Protein similarities of SAGA encoding gene in Arabidopsis, O. sativa, human and S. cerevisiae.

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S3 Table. List of primer used in qRT-PCR.

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S4 Table. Gene Ontology list of Arabidopsis and O. sativa for SAGA complex gene.

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S5 Table. List of Arabidopsis SAGA complex subunits known and predicted protein interactions from STRING database.

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S6 Table. List of S. cerevisiae SAGA complex subunits known and predicted protein interactions from STRING database.

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S7 Table. MPSS data for Arabidopsis SAGA complex encoding genes showing different tissue-specific abundance.

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S8 Table. MPSS data for O. sativa SAGA complex encoding genes showing different tissue-specific abundance.

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S9 Table. List of co-expression genes in Arabidopsis.

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S10 Table. List of genes obtained from ATTED-II for Arabidopsis SAGA complex co-expressed gene network analysis.

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S11 Table. Analysis of cis-regulatory element in 1000bp upstream promoter sequences from TSS in SAGA complex subunit genes using PlantCARE and PLACE database.

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.


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