Interacting partners of Brassica juncea regulator of G-protein signaling protein suggest its role in cell wall metabolism and cellular signaling

Heterotrimeric G-protein (hereafter G-protein) signaling plays a pivotal role in regulating various biological and cellular functions across phyla [1,2]. The core G-protein complex consists of Gα, Gβ, and Gγ subunits. In metazoans, ligand binding to G-protein coupled receptor (GPCR) facilitates the exchange of GTP for GDP on the Gα subunit, thereby dissociating the inactive core heterotrimer (Gαβy) complex into Gα-GTP and Gβy dimer [3,4]. Both these components interact separately with their effector proteins to regulate diverse downstream signaling pathways. Hydrolysis of Gα-GTP to Gα-GDP by the intrinsic GTP hydrolysis activity of Gα allows the reformation of the inactive heterotrimeric complex leading to the termination of the signal [5]. In addition, GTPase-accelerating activity of Regulator of G-protein Signaling (RGS) proteins further enhances the Gα-GTP hydrolysis, thereby desensitizing the G-protein-mediated signaling [6].

Although the core subunits of the G-protein complex remain the same across phyla, plants are known to possess unique regulation of G-protein signaling activation and deactivation. In the absence of typical GPCRs in plants, the activation of G-protein signaling relies primarily on the self-activating property of the Gα subunit. Additionally, in the plant kingdom, RGS protein acts as a crucial regulatory component that deactivates the G-protein signaling [2]. Plant RGS protein contains the N-terminal located ‘seven transmembrane (7-TM) structure’, which is absent from its animal counterparts, and the C-terminus cytoplasmic ‘RGS box’, which shows a high level of sequence similarity with archetypal RGS proteins [7]. The C-terminal located ‘RGS-box’ of plant RGS possesses the GAP activity [8–11].

Previous studies in Arabidopsis show the importance of sole RGS protein in regulating various plant growth and developmental processes like cell proliferation, germination, stomatal movement, sugar sensing, and response to phytohormone and various environmental cues [8,12–15]. The Arabidopsis rgs1 mutant shows hyperactive responses like an increase in leaf and hypocotyl length. Ectopic expression of AtRGS1 confers smaller rosette size and shorter hypocotyl length [13]. Suppression of RGS protein leads to hyposensitive stress responses in Arabidopsis and mulberry [15,16]. In recent decades, tremendous progress has been made in elucidating the physiological responses and developmental phenotypes of plant G-protein components; however, the intricacies of the molecular cascade associated with these responses are yet to be discovered.

In a multicellular system, protein–protein interactions (PPIs) play a very crucial role in regulating various cellular processes. Yeast-two-hybrid (Y2H) has been widely used for the identification of the interacting partners of proteins associated with complex regulatory pathways [17–19]. Compared with metazoans, limited sets of effector molecules for G-protein components have been identified from plants, particularly from crop species, and their characterization is still in the infancy stage. In an earlier study using Y2H and proteomic-based screening approaches, various targets of core G-protein components have been reported from the model plant Arabidopsis [17,18,20]. Recently, Y2H screening of multiple Gβ subunits (paralogs) of Brassica juncea led to the identification of both subunit-specific and common interacting partners, that are known to control a wide range of cellular and biological processes [21].

B. juncea (AABB, 2n=36) is a natural interspecific hybrid between Brassica rapa (AA, 2n=20) and Brassica nigra (BB, 2n=16) [22,23]. It is grown worldwide as an important oilseed, vegetable, and condiment crop. Evolutionary events like whole-genome triplication (WGT), allopolyploidization, and genomic rearrangements in Brassica lineage have created multiple homeologs shaping tremendous morphological, developmental, and chemical plasticity across Brassica crop species [24,25]. Since G-protein signaling components control various aspects of plant growth and development [2], identification of the G-protein-effectors could be crucial to outlining the regulatory networks involved in multiple biological and cellular functions.

In the present study, we identified multiple homologs of RGS proteins from the allotetraploid B. juncea. Furthermore, a well-expressed B. juncea RGS isoform was selected as bait protein to screen the Y2H cDNA library constructed from mRNA isolated from reproductive tissues of B. juncea. A large number of RGS-interacting proteins were identified, and Gene Ontology (GO) analysis predicted their involvement in a wide range of biological and molecular functions. Furthermore, coexpression analysis of the RGS-interacting partners is also presented. These findings increase our knowledge of the RGS-interacting proteins and provide a resource for developing a plant G-protein interactome network.

In the present study, B. juncea L. (cv. Varuna) was grown under short-day conditions (10 h light/14 h dark) at 24°C with a photon flux density of approximately 300 μmol m⁻² s⁻¹ and 60–70% relative humidity. Tissue types representing different developmental stages like 5-day-old seedlings, root, stem, young leaves from 1-month-old plants, flower (unopen flower buds), and developing siliques (7 and 14 days-after-pollination (DAP)) were collected and stored at −80°C.

Gene-specific primers (Supplementary Table S1) were designed based on our previously reported RGS sequences from Brassica species [11], and the full-length RGS coding DNA sequences (CDS) were amplified from B. juncea. Following PCR amplification, the PCR products were cloned into a pENTR/D-TOPO vector (Invitrogen) and sequenced to determine the accuracy and identity of the clones. Phylogenetic analysis was carried out using deduced RGS sequences of B. juncea and those retrieved from the order Brassicales (https://phytozome.jgi.doe.gov/pz/portal.html) using the neighbor-joining method with 1000 bootstrap iterations in MEGAX-32 [26].

Total RNA isolation was from different vegetative and reproductive developmental tissues (seedling, root, leaves, stem, flower, siliques) of B. juncea. The first-strand cDNA synthesis and qRT-PCR were conducted in the same manner as previously described [27]. The cDNA samples representing different stages of plant growth and development were diluted to 1:20 and qRT-PCR was performed using gene-specific primers (Supplementary Table S1).

To construct a bait vector for the Y2H analysis, the CDS of BjuA.RGS1box along with C-terminal (BjuA.RGS1box+Ct domain) was amplified with primers containing the restriction sites of NcoI and EcoRI (Supplementary Table S1) and then cloned into bait vector ‘pGBKT7’ harboring GAL4 DNA-binding domain (BD). Subsequently, the bait vector pGBKT7-BjuA.RGS1box+Ct domain was introduced into yeast strain Y2H-Gold using PEG/LiAc-mediated yeast transformation and plated on a minimal medium lacking tryptophan (SD/−Trp). Furthermore, autoactivation of pGBKT7-BjuA.RGS1box+Ct domain bait construct was tested on SD/−Trp plates supplemented with X-α-Gal (40 mg ml⁻¹) and Aureobasidin A (125 ng ml⁻¹).

The construction of B. juncea Y2H library used in the present study was previously reported [21], and was developed from the RNA isolated from reproductive stages (flowers and developing siliques) of B. juncea. For screening the interacting proteins of BjuA.RGS1, the bait strain cells containing the pGBKT7-BjuA.RGS1box+Ct domain were mated with B. juncea prey library cells in 2X YPDA liquid medium and incubated for 28 h at 30°C. The mated culture was plated onto SD/-Leu/-Trp (double drop-out (DDO)) plates containing X-α-Gal (40 mg ml⁻¹) and Aureobasidin A (125 ng ml⁻¹) and kept at 30°C for 5 days. Moreover, 100 µl of the mated culture was also plated on SD/-Leu and SD/-Lue/-Trp medium in different dilutions (1/10, 1/100, 1/1000, and 1/10000), and mating efficiency was calculated by the number of CFU/ml of diploids divided by CFU/ml of limiting factor (prey libraries) × 100 on SD/-Leu plate. Furthermore, colonies grown for 4–5 days (blue colonies) obtained on DDO/X/A were streaked onto the higher stringent medium SD/-Leu/-Trp/-Ade/-His (quadruple drop out (QDO)) containing X-α-Gal (40 mg ml⁻¹) and Aureobasidin A (125 ng ml⁻¹) (QDO/X/A).

Furthermore, yeast colony PCR was performed on colonies showing activation of all the reporter genes and PCR products were sequenced. To identify the interacting proteins, sequences were analyzed using BLAST analysis in BRAD (http://brassicadb.org/brad/blastPage.php) and NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) databases. Furthermore, functional annotation of the identified BjuA.RGS1-interacting proteins was obtained after GO analysis using PlantGSEA (Plant GeneSet Enrichment Analysis) with A. thaliana used as the background genome [28]. The GO terms enrichments were carried out using Fisher’s test and Benjamini–Hochberg (false-discovery rate cutoff of 0.05) correction applied for calculation of adjusted P-values. Thereafter, REVIGO was used to visualize the interactive graph of over-represented GO terms [29]

In the present study, four full-length RGS homologs (BjuA.RGS1, BjuA.RGS2, BjuB.RGS1, and BjuB.RGS2) like sequences were isolated from B. juncea and cloned into pENTR/D-TOPO entry vector. The B. juncea RGS homologs were classified and named based on the sequences obtained from its progenitor genomes, i.e. B. rapa and B. nigra. The BjuA.RGS1 and BjuA.RGS2 corresponds to A-genome-specific BraA.RGS1 and BjuA.RGS2, respectively; whereas BjuB.RGS1 and BjuB.RGS2 correspond to BniB.RGS1 and BniB.RGS2, respectively. Full-length coding RGS sequences isolated from B. juncea ranged from 1368 to 1386 bp, encoding proteins of 455–461 amino acids in length. Deduced RGS proteins of B. juncea shared 84.6–95.2% identity among themselves and 84–88.9% identity with the A. thaliana AtRGS1 (Supplementary Table S2). Amino acid sequence alignment showed that the B. juncea RGS proteins contain the N-terminal ‘7-TM domain’ and the C-terminal located ‘RGS domain’, and share high sequence conservation with orthologs from B. rapa and B. nigra (Figure 1A) [11]. Furthermore, a key residue (Glu320) responsible for the GAP activity of Arabidopsis RGS protein was also found to be highly conserved in BjuRGS proteins [9]. Phylogenetic analysis of RGS proteins belonging to core Brassicaceae revealed that the BjuRGS proteins grouped into two independent orthologous clades, one containing the RGS1 and other RGS2 proteins (Figure 1B).

To get the primary insight into the role of multiple RGS homologs in B. juncea, we further determined the expression level of BjuRGS genes across various stages of plant growth and development. The qRT-PCR analysis showed that all the members of B. juncea RGS genes were expressed although showing differential expression patterns (Figure 1C). The BjuA.RGS1 and BjuB.RGS1 were found to be highly expressed genes with profound expression observed in the root, flower, and siliques stages as also reported for the B. rapa ortholog, BraA.RGS1 [11]. The BjuB.RGS2 transcript had low abundance across all the examined tissue types. Structural and gene expression changes are an important characteristic of polyploidy, and duplicated genes, i.e. homeologs often tend to possess differential expression patterns [30,31]. B. juncea is an allopolyploid, formed by the fusion of two different genomes. In the present study, the differential expression pattern was observed for the BjuRGS homologs across different developmental stages. Interestingly, the expression of type-I (BjuRGS1) homologs that shared the close phylogeny with Arabidopsis counterpart (AtRGS1) was predominantly higher than BjuRGS2 homologs (Figure 1B). Overall, the expression data suggest the possible functional dominance of type-I RGS homeologs in the allopolyploid B. juncea. The expression dominance of G-protein gene homeologs is quite a norm in polyploid plant species like soybean and Brassica, and this phenomenon is known to regulate the G-protein activities and biological functions [10,11,21,32].

It is well established that only the C-terminal region (Ct) of RGS protein possesses the GAP [8,9]. Therefore, to carry out the Y2H screening, the Ct of the ubiquitously expressed group-I RGS protein, i.e. BjuA.RGS1 was selected for the screening. The BjuA.RGS1box including its Ct was cloned into a pGBKT7 bait vector. The yeast cells containing bait clone alone were unable to grow on the selection plate (DDO/X/A), indicating that BjuA.RGS1box+Ct domain does not display autoactivation property. Further mating efficiency was determined as 5.3%, which was within the range of 2–5%, as per the manufacturer’s protocol. A total of approximately 2000 blue colonies were obtained on the double drop-out selection medium (DDO/X/A) (Table 1). Furthermore, these colonies were patched on a more stringent quadruple drop out (QDO/X/A) minimal medium, which resulted in optimum growth of more than 1900 colonies.

Furthermore, colony PCR was performed using primers for the pGAL4-AD prey vector (Supplementary Table S1), and a total of 1000 blue clones were sequenced to reveal their identity. Sequence analyses using BLASTn revealed 51 nonredundant clones as the putative-interacting partners of BjuA.RGS1 protein. To rule out autoactivation and re-examine the interaction, rescued prey plasmids were individually cotransformed into yeast strain Y2H-Gold along with bait (pGBKT7-BjuA.RGS1box+Ct domain) or empty vector (pGBKT7). The one-to-one Y2H assay further confirmed 30 out of 51 prey plasmids to be genuine interactors of BjuA.RGS1box+Ct domain (Figure 2), while the remaining 21 prey plasmids either displayed autoactivation or were not in-frame (Table 1). Furthermore, to validate the positive and genuine interaction, a one-to-one Y2H assay was performed using 15 prey plasmids (representing various important biological functions) cotransformed with pGBKT7-BjuA.RGS1 box+Ct domain or pGBKT7 empty vector into Y2HGold strain (Supplementary Figure S1). The selection was carried out on growing diploid yeast cells on a QDO medium containing different concentrations of 3-Amino-1.2.4-triazole (3-AT). The 3-AT is a competitive inhibitor of HIS3 gene product used as a reporter in Y2H and is typically added to select the strong interactions and eliminate the false-positive results. The tested combination shows growth on the QDO+3-AT medium, therefore, ruling out the possibility of false-negative interaction. Overall, several of the identified BjuA.RGS1box+Ct domain-interacting proteins showed a differential level of interaction strength, as noted by growth and blue color intensity on different selection mediums (Figure 1 and Supplementary Figure S1).

As the information about the genomic resources for Brassica species is limited, functional annotation of the interacting proteins identified for BjuA.RGS1box+Ct domain was performed using the GO tool of the TAIR database. The interacting proteins identified in the present study are novel targets of plant RGS, showing involvement in various biological and cellular functions (Table 2). Furthermore, functional enrichment analysis was also carried out using the PlantGSEA using A. thaliana whole genome annotation as the background. TAIR gene IDs were submitted to PlantGSEA and GO terms with their associated significance values (P-values) were obtained (Supplementary Table S3). REVIGO visualization of over-represented GO terms grouped the interacting proteins based on their biological process, molecular function, and cellular component (Supplementary Figure S2). In the biological process, the most enriched GO terms are associated with BraA.RGS1-interacting proteins were metabolism, ion transport, and cytoskeleton organization. In terms of molecular function, GO terms were associated with nucleotide and amino acid binding, kinase activity, oxidoreductase activity, α-mannosidase activity, and transporter activity. The cellular compartment category revealed the distribution of proteins into various cellular and subcellular categories, namely membrane, cytosol, cell wall, cell junction, and plastid.

A more in-depth analysis revealed that the majority of the interacting proteins are involved in the biosynthetic processes, which indicate the important role of BjuA.RGS1 in plant metabolism (Table 2). The metabolic changes in the cell wall play an important role in plant development, particularly during fruit ripening and silique maturation [33,34]. These changes are mediated by various cell wall synthesis, loosening, and degrading enzymes, which control the overall dynamics of fruit and silique maturation and dehiscence. In our study, multiple cell wall synthesis and degrading enzymes like BjuEXP6, Bju-α-MAN, BjuPGU4, and BjuRMS3, were identified as the interacting partner for BjuA.RGS1box+Ct domain. Interaction of these proteins could possibly activate the heterotrimeric G-protein signaling and their associated downstream components, which eventually regulate the cell wall metabolism during the pod maturation stage in Brassica species. Earlier, Klopffleisch et al. [17] through Y2H library screening also identified various interacting partners of G-protein components that are involved in cell wall metabolism. Besides, the identification of interacting proteins like BjuGGDR, BjuASP2, BjuGLN1, and BjuSAsoMet2 in the current study suggests the probable involvement of the identified BjuRGS1-targets in other biosynthetic processes of B. juncea.

Furthermore, various kinases like BjuMEK4, BjuDGK3, and serine/threonine-protein kinase were also found to interact with the BjuA.RGS1box+Ct domain (Table 2). In plants, phosphorylation of RGS protein by RLKs, WNK, and other kinases are likely to activate the downstream G-protein signaling [14,35–38]. The protein kinases identified in the present study could play an important role in the phosphorylation of BjuA.RGS1, resulting in its separation from cognate Gα protein, thereby reinforcing phosphorylation-based activation and deactivation regulation of the G-protein cycle in plants, which needs further investigation.

The proteins that are interacting with each other could possess similar biological functions, and cellular localization, and are more likely to be coexpressed [39]. To investigate the coexpression pattern, we analyzed that the expression of selected BjuA.RGS1-interacting proteins represents various classes of biological functions, in different tissue types of B. juncea (Figure 3). The BjuEF1B2, BjuExp6, BjuAIP, BjuGGDR, and BjuSAdoMet2 genes show profound expression in different tissue types. Besides, a few interacting genes like BjuKinase, BjuASP2, BjuPGD1, and BjuDGK3 were found to be predominantly expressed during flowering and silique stages, suggesting their possible involvement with BjuRGS proteins in plant reproduction.

In summary, we identified multiple RGS-encoding genes in the allotetraploid B. juncea, displaying distinct transcriptional differentiation during plant developmental stages. Using the Y2H approach, a total of 30 BjuA.RGS1-interacting proteins were identified that are involved in diverse biological and cellular functions, and primarily associated with biosynthetic and signaling transduction processes. Furthermore, in-depth characterization of these interacting proteins will help in establishing their role in RGS-dependent and independent G-protein signaling processes specific to plant lineage.

Data associated with the paper can be accessed by contacting the authors.

The authors declare that they have no known competing financial interests that could have appeared to influence the work reported in the present paper.

The present work was supported by funding from SERB, India (grant no. EMR/2016/006433) to N.C.B. R.K. was funded by the UGC (India) and NIPGR short-term research fellowship.

Roshan Kumar: Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review & editing. Naveen C. Bisht: Conceptualization, Resources, Data curation, Software, Supervision, Funding acquisition, Validation, Investigation, Visualization, Project administration, Writing—review & editing.

The central instrumentation facility and plant growth facility are acknowledged.

3-AT

3-Amino-1.2.4-triazole

7-TM

seven transmembrane

BD

binding domain

BRAD

Brassicaceae Database

CDS

coding DNA sequences

CFU

colony-forming unit

Ct

C-terminal region

DAP

days-after-pollination

DDO

double drop-out

GO

gene ontology

GPCR

G-protein coupled receptor

NCBI

National Center for Biotechnology Information

PlantGSEA

Plant GeneSet Enrichment Analysis

PPI

protein–protein interaction

QDO

quadruple drop out

RGS

Regulator of G-protein Signaling

TAIR

The Arabidopsis Information Resource

Trp

tryptophan

WGT

whole-genome triplication

Y2H

Yeast-two-hybrid