Stress induced β subunit of heterotrimeric G-proteins from Pisum sativum interacts with mitogen activated protein kinase

Abstract

We here report in Pisum sativum system a novel protein-protein interaction of β-subunit of heterotrimeric G-proteins (PsGβ) with a Mitogen activated protein kinase (PsMPK3) during cDNA library screening by yeast-two-hybrid assay. The transcript of these two genes also showed co-regulation under abscisic acid (ABA) and methyl jasmonate (MeJA) treatments. The protein-protein interaction was further validated by performing one-to-one interaction and β-galactosidase assay in yeast system. β-subunit of G-proteins from a heterologous system Oryzae sativa also showed interaction with PsMPK3. The interaction between PsGβ and PsMPK3 was further confirmed by in vitro protein-protein interaction. This suggested that MPK3 function as effector molecule for Gβ, which may helps in the regulation of stomatal functioning. These findings also provide an evidence for a possible cross-talk between MPK3 and G-protein-mediated signaling pathways in plants.

Introduction

Abiotic and biotic stresses are the major constraints of modern day agriculture and they not only affect the growth of plants, but also penalize the productivity of the crop.^1,2 Recently with the discovery of genetic tools and stress responsive genes it has become easy to develop stress tolerant crops for the sustainable agriculture.³ G-protein-coupled receptors (GPCR) and G-proteins are some of those components that play direct role in abiotic and biotic stress signaling.^4,5 There are many stimuli that become the basis of various responses in the plants ranging from light, various chemicals, diverse abiotic stresses, etc. The stimuli are perceived at the membrane level and with the help of various other cytosolic proteins they are transduced to the nucleus for translating it into a response. The proteins that take part in this mechanism are called secondary messengers or signaling molecules. Many proteins and molecules have so far been characterized, G-proteins being one of them. The objective of the present study is to link the signals that are perceived at the membrane level and transferred to the nucleus with the help of various unknown effectors and secondary messengers.

It has been reported that overexpression of beta subunit of G-proteins (Gβ) gave heat tolerance to the tobacco plants. Interestingly, involvement of MAP kinase a component of phospho-relay signaling cascade in G-proteins signaling through Gβ subunit is already reported in Saccharomyces cerevisiae.⁶ The activation of MAP kinase pathway is through heterotrimeric G-proteins, which in turn were activated by pheromones.^7–9 Gβ also interacts with the non-catalytic carboxy-terminal regions of Ste20 and its mammalian homologues, the p21-activated protein kinases (PAKs) in the same organism.¹⁰ The Gβ subunits possess WD40 domain, which has made it a favorite among the proteins that can interact with other proteins to carry out significant cellular processes. The WD40 domain is a motif that contains 40–43 amino acids in the beta subunit of G-proteins.¹¹ It acts like a circular propeller with a pore in center.^12,13 The seven blades of the propeller have a binding potential where other proteins or small ligands can bind.¹⁴ Since G-proteins effectors in plants are scarce, the signaling through them is still unknown. The role of G-proteins in regulating ion channels in animals as well as plants is already known.^15,16 It also controls the development of roots, leaves and floral parts in plants.^17–19 Gβ subunits are also known to regulate the action of K⁺ channels in stomata.¹⁵

Mitogen activated protein kinase (MAPK) cascade is one of the most conserved signaling cascade across the kingdom and is involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment.^6,20 This cascade consists essentially of three components, an MAPK kinase kinase (MAPKKK), an MAPK kinase (MAPKK) and an MAPK connected to each other by the event of phosphorylation. Signaling through MAP kinase cascade can lead to cellular responses including cell division, differentiation as well as responses to various stresses. One of the Arabidopsis MAP kinase, AtMPK3, is shown to be involved in stomata functioning²¹ which is also an intrinsic mechanism resistance against fungal and bacterial pathogen.¹⁶

We here report interaction of β-subunit of G protein (PsGβ) and a mitogen activated protein kinase (PsMPK3) in Pisum sativum. We extended this finding in rice and showed the interaction of rice OsGβ with PsMPK3 and OsMPK3. The implication of this interaction has been discussed in a hypothetical model.

Results

Cloning of β subunit of G protein (PsGβ) from Pisum sativum.

The cDNA clone of PsGβ was obtained after screening the pea cDNA library using radio labeled tobacco Gβ cDNA as a probe. Sequence analysis of PsGβ cDNA shows that it is a full-length cDNA, which is 1.13 kb in size and encodes a protein consisting of 377 amino acid residues with a predicted molecular mass of about 35 kDa and pI 7.04. Accession number of Pisum sativum PsGβ is AF145976.

Screening of interacting partners of Pisum sativum Gβ using yeast-two-hybrid assay.

For the yeast-two-hybrid assay Gal4-based two-hybrid system from Clontech, USA was used. Pisum sativum cDNA library cloned in yeast pGADT7 vector was used as a prey and Pisum sativum Gβ cloned in pGBKT7 was used as bait. Both genes were co-transformed into yeast strain AH109 and growth of five colonies was observed in 3 DO-SD medium (for detail please see Materials and Methods). The colony number II showed much better growth than the others (Fig. 2A-II and upper part). The β-galactosidase assay was performed for all the five colonies and the result showed that the colony number II represents the strongest interaction (Fig. 2A). Therefore, the colony II was sequenced and the analysis showed that this is a mitogen activated protein kinase 3 (PsMPK3; accession number AF153061).

Figure 2.

(A) Comparative analysis of interacting partners of PsGβ protein. Five interacting partners out of which one grew better (colony II) on SD lacking LTH⁺ 20 mM 3AT is finally selected for sequencing and for further study. The upper panel shows the growth of colonies and the β-galactosidase filter lift assay of the same colonies. Lower part is quantitative data. (B)Yeast two-hybrid system-based interaction between Gβ and MPK3: (I) template for parts (II–VI), showing phenotypes on (II) a YPD plate, (III) on a synthetic dextrose plate lacking tryptophan, (IV) on synthetic dextrose plate lacking leucine, (V) on a synthetic dextrose plate lacking leucine, tryptophan and histidine and (VI) β-galactosidase filter lift assay. PsGβ and PsGα is a positive control (1), Clone #1 is PsGα + PsMPK3 (2), Clone #2 is OsGβ + PsMPK3 (3), Clone #3 is OsGβ + OsMPK3 (4) AD + BD is a negative control (8) AH109 is a host control (6), AD is a single vector control (7) and BD is also single vector control (5). (C) TNT^® High-Yield Reactions were performed with 35S-radiolabelled methionine in vitro Translation Labeling System. Lanes 1–3 are 5 ml of TNT reactions expressing each of the following: lane 1 is a positive control (co-immunoprecipitation of PsGβ and PsGα); lanes 2 and 3 are co-immunoprecipitation of PsGβ and PsMPK3 (experiment).

PsGβ and PsMPK3 transcripts showed co-regulation under ABA and MeJA treatments.

We further analyzed the transcript accumulation of PsGβ and PsMPK3 under the treatment of hormones known to play roles in various stresses, abscisic acid (ABA) and methyl jasmonate (MeJA). Pea seedlings that were 7 to 10 days old were exposed to various concentrations of ABA and MeJA (5, 50 and 100 µM) for a period of up to 24 h (0, 2, 4, 8, 12 and 24 h). Semi-quantitative RT-PCR analysis revealed significant induction of PsGβ and PsMPK3 mRNAs after 4 h exposure to various concentrations of ABA and MeJA (Fig. 1A and B). The result obtained by semi-quantitative RT-PCR was further validated by quantitative real time PCR (qRT-PCR) after 2, 4 and 8 h exposure of 100 µM of both ABA and MeJA (Fig. 1C and D). Total RNA was isolated from 10-day-old seedlings treated with ABA and MeJA, reverse transcribed and used for real-time PCR in the presence of SYBR-Green intercalating dye. Amplification of the ACTIN gene under identical conditions served as an internal control. Both PsGβ and PsMPK3 gene showed co-upregulation compared to the internal control (Fig. 1C and D).

Figure 1.

Semi quantitative RT-PCR analysis of the transcripts of PsGβ and PsMPK3 genes in the presence of (A) ABA and (B) MeJA for a period of 24 h. Quantitative real time PCR (qRT-PCR) analysis at 2, 8 and 24 h post-100 µM (C) ABA and (D) MeJA treatments. Actin gene is used as an internal control for both the experiments. The experiments were repeated three times with similar results.

β-subunit of G-proteins interacts with MPK3 in pea and rice.

For validating the interaction of PsGβ and PsMPK3, one-to-one interaction of the two proteins was carried out using yeast-two-hybrid assay. The complete ORF of PsGβ gene was cloned in a yeast AD vector (pGADT7) while the complete ORF of PsMPK3 gene was cloned in yeast BD vector (pGBKT7). Figure 2B(I) is a template for (II–VI) of Figure 2B, showing the streaked clones. AD-PsGβ and BD-PsMPK3 was co-transformed in yeast AH109 cells (Fig. 2B(II)) and then screened on single (Trp⁻ and Leu⁻) and triple [(Leu⁻, Trp⁻ and His⁻ (-LTH) + 20 mM 3-AT (3-Amino-1,2,4-triazole)] DO⁻SD media [Fig. 2B(III–V), respectively]. The interaction was also confirmed through β-galactosidase assay [Fig. 2B(VI)]. We further investigated the interaction of β-subunit of G proteins with MAP kinase by taking a heterologous system, Oryzae sativa. The β-subunit of G proteins (OsGβ) and MAP kinase (OsMPK3, Acc. No. DQ826422.1) from rice were cloned by designing primers against the sequences available in the database. The genes were further cloned in-frame into yeast vectors for interaction analysis. OsGβ was cloned in AD vector (pGADT7) while OsMPK3 was cloned in BD vector (PGBKT7). To test the interaction between heterologous system AD-OsGβ was co-transformed with BD-PsMPK3 and screened on the similar selection conditions, the interaction was confirmed by β-galactosidase assay [Fig. 2B(VI)]. The interaction of OsGβ and OsMPK3 was also confirmed by co-transforming yeast AH109 with AD-OsGβ and BD-OsMPK3 (Fig. 2B(I–VI)). As a positive control we used BD-PsGβ and AD-PsGα,⁵ while empty vectors co-transformed in AH109 served as negative control. The results show that all the colonies grew in AH109 (Fig. 2B-II), whereas colonies 6 (AH109 host cells) and 7 that contain yeast cell harboring empty AD vector (pGADT7) did not grow in Trp⁻ [Fig. 2B(III)] and colony 5 that contains BD vector (PGBKT7) did not grow in Leu⁻ [Fig. 2B(IV)]. All the experimental clones [(Clone #1: PsGβ + PsMPK3 (2), Clone #2: OsGβ + PsMPK3 (3) and Clone #3: OsGβ + OsMPK3 (4)] along with positive control (1) grew in -LTH + 20 mM 3-AT [Fig. 2B(V)].

The interaction between PsGβ and PsMPK3 was further confirmed by in vitro protein-protein interaction by using co-immunoprecipitation. The detail procedure is described in Materials and Methods. The results of the interaction of PsGβ and PsMPK3 by co-immunoprecipitation with anti-PsGβ antibodies are shown in Figure 2C. The results show that PsGβ interacted with PsMPK3 protein (Fig. 2C and lanes 2 and 3) as well as with PsGa as positive control (Fig. 2C and lane 1). These results further confirmed that these proteins interact with each other.

Discussion

We here report a novel interaction of Gβ subunit with MPK3 in Pisum sativum through yeast-two-hybrid assay. Interestingly, this interaction holds true in Oryzae sativa system too. Additionally, the transcripts of PsGβ and PsMPK3 showed co-regulation under the treatment of plant growth hormone like ABA and MeJA. G-proteins are smart small molecular protein particle that are capable of interacting with other proteins to activate various actions, which are required for the normal functioning of cell under adverse conditions.

Beta and γ subunits of G protein are significant for the growth and development of plant.²⁴ GCR1 mutant like agb1 and gpa1 mutants is hypersensitive to ABA during germination.⁴ Among the three mutants, Arabidopsis agb1 is predominant regulator of ABA signaling and it could work in tandem with other effectors. Gβ subunit is involved in almost all the major decisive role of plant growth and development.²⁴ It is involved in maintaining shape of leaves, stomatal regulation and root development. Loss of function in AGB1 gene causes change in shape of leaves and flower structure.¹⁹ AGB1 is expressed more in roots causing longer primary root and more lateral roots^17,25 and recently it was shown that AGB1 bind with NLD1(N-MYC DOWNREGULATED-LIKE1) protein and regulates the root architecture.²⁶ Functions of AGB1 is not only restricted to roots but they controls the opening and closing of stomata²⁷ and protect the leaves from oxidative stress.²⁸ The mechanisms of all these findings are yet to be elucidated.

Since MPK3, a member of MAPK family known to play significant role in plant defense machinery, we hypothesize that interaction of Gβ subunit with MPK3 might have its role in stomatal movement (Fig. 3). The MAP kinase class of proteins belongs to a large family of serine threonine protein kinases. They are basically located in the cytoplasm part of cell and upon activation get translocated to the nucleus for activating transcription factors thus bringing about the upregulation of genes.²⁰ Some MAPK can remain in cytoplasm and they function by interacting with other protein bringing about phosphorylation and modulation. Mutant of G-proteins and MAPK are compromised for resistance and plant become susceptible to pathogen, except gpa1 mutant of Arabidopsis, which is resistant to pathogens. G-proteins and MAP kinases are linked to ABA signaling. ABA also regulates the functioning of stomata by some unknown mechanisms. Based on our finding of interaction of Gβ subunit of G-proteins with MPK3 in pea and rice and the co-regulation of their transcript in pea we postulated a hypothetical model showing the role of these two proteins in stomatal movement (Fig. 3). In the model we also included the role of β-subunit of G-proteins in other biological functions as mentioned above and in the legend of the figure. Though it will be interesting to elucidate the biological significance of the interaction of β-subunit of G-proteins with MPK3 in plant system. Overall, the discovery of the cross-talk between Gβ and MPK3 makes an important contribution to our better understanding of G-protein mediated signaling pathways and abiotic stress signaling in plants.

Figure 3.

The proposed model depicts the role of probable interaction between Gβ subunit with MPK3 and cross talk between various signaling molecules. Gβ subunit of G-proteins act as a downstream effectors that interacts with several others like NLD1 (N-MYC DOWNREGULATED-LIKE1),²⁶ and XLG2 (Extra Large G-Protein 2),²⁹ whereas its natural interacting partner Gα interacts with PLC (PHOSPHOLIPASE C),⁵ THF1 (THYLAKOID FORMATION 1),³⁰ AtPirin³¹ and PD1 (Prephenate dehydratase 1).³² The OST1 (OPEN STOMATA 1) is ABA-activated protein kinase; OST1 acts in the interval between ABA perception and ROS production.³³ The ROS (reactive oxygen species) H₂O₂ and ABA in turn activate MPK3. NO (nitric oxide) also activate MPK3.¹⁶ Which in turn is activated by calcium reserve. MPK3 is reported to regulate the stomatal functioning.²¹

Material and Methods

Construction of P. sativum cDNA library.

A cDNA library was constructed from 5 µg of poly(A) RNA (isolated from the top four leaves of 7-day-old pea seedlings) in Uni-Zap XR vector using a Zap cDNA synthesis kit (Stratagene; http://www.stratagene.com/) according to the manufacturer's protocol. The resulting phage library contained 1 × 10⁹ plaque-forming units per ml.

Cloning of cDNAs of G-protein β subunit from pea.

For cloning of the Gβ subunit, the pea cDNA library was screened using a radiolabeled tobacco Gβ subunit as probe, kindly provided by Dr. R. Oelmuller (Ludwig-Maximilians-Universitat, Jena, Germany). Various clones were obtained after tertiary screening of the library. cDNA sequencing was done by Macrogen, Korea (www.macrogen.com/eng/sequencing/dua.jsp) using Sanger's method. Most of the routine sequence (DNA and amino acid) analysis was performed using MACVECTOR (version 7; Oxford Molecular Group). A homology search was performed using BLAST (NCBI), (www.ncbi.nlm.nih.gov/BLAST).

Yeast-two-hybrid assay to study the interaction of proteins.

A Gal4-based two-hybrid system was used as described by the manufacturer (Clontech; www.clontech.com/). The coding region of the Gβ subunit (1,134 bp) was amplified by PCR with primers harboring restriction sites, and cloned in-frame into the EcoRI and BglII sites of the binding domain vector pGBKT7. This resulted in the vector pGBKT7-PsGβ was co-transformed with Pisum sativum AD library into yeast strain AH109 harboring two reporter genes (HIS3 and β-galactosidase) by the lithium acetate method. AH109 contains integrated copies of ADE2, HIS3 and lacZ (MAL1) reporter genes under the control of distinct GAL4 upstream activating sequences (UAS) and TATA box. These promoters yield strong and very specific responses to GAL4. Yeast cells carrying both the plasmids were selected on the synthetic medium lacking Leu and Trp (SD-Leu-Trp⁻). The yeast cells were then streaked onto a SD medium [(Leu), (Trp), (His)] containing 20 mM 3-AT (3-Amino-1,2,4-triazole) to determine expression of the HIS3 nutritional reporter. The β-galactosidase expression of the His⁺ colonies was analyzed by filter-lift assays as described by the manufacturer (Clontech). The resulted positive clones including PsMPK3 was cloned in pGEMT easy vector promega and further it was cloned in pGBKT7 vector at Nde1 and BamH1 sites, the resulted clone was co-transformed in AH109 and was assayed on dropouts medias and also by β-galactosidase assay.

In vitro interaction by protein expression and co-immunoprecipitation.

For optimal expression in wheat germ extracts, the open reading frame of PsGβ, PsGα and PsMPK3 were cloned into the pGEMT easy vector (Promega). The TNT^® Coupled Wheat Germ Extract System (Promega) is used to express the recombinant proteins. TNT^® reactions were assembled following the manufacturer's protocol. Reactions were incubated at 25°C for 2 h. Prior to loading on a gel, reactions were treated with RNase ONE™ Ribonuclease to remove unincorporated radiolabelled charged tRNA (1 unit/ml, 5 min at 37°C). Immunoprecipitations were carried out using the following protocol. All incubations were at room temperature with end-over-end rotation. Ten µl of each TNT^® reaction (PsGβ and PsGα or PsGβ and PsMPK3) were mixed with 50 µl immunoprecipitation buffer (20 mM Tris (pH 7.5) + 150 mM NaCl + 0.2% Triton X-100) and incubated for 1 h followed by following steps:

1. 0.6 mg anti-PsGβ rabbit polyclonal antibody was added to each reaction and incubated 1 h.

2. 5 µl Protein A agarose (Sigma, cat.#P7786) was added to each reaction and incubated 1 h.

3. Immunoprecipitates were collected by centrifugation at 7,000x g for 10 sec. The supernatant was carefully removed, and immunoprecipitates were washed by resuspending in 0.5 ml immunoprecipitation buffer. This was repeated for a total of fourwashes.

4. The final pellet was suspended in 20 µl 1x SDS sample buffer, heated at 70°C for 3 min, centrifuged briefly and 20 µl was loaded.

TNT^® reactions and immunoprecipitation supernatants were run on 10% SDS PAGE radiolabeled protein was detected using GE Amersham phosphoimmager.

ABA and MeJA application.

Seven-day-old pea seedlings were transferred into media containing ABA and MeJA (5 µM, 50 µM and 100 µM) for a period of 24 h. Young leaves of the stressed seedlings were harvested after the indicated time period. Seedlings grown in water were used as a control.

Expression studies by semi quantitative PCR and quantitative real time-PCR (qRT-PCR).

To study the expression of PsGβ and PsMPK3, leaf tissue of various stress treated leaves were harvested for RNA isolation, ground in liquid nitrogen and stored at −80°C. Total RNA was isolated with the Qiagen RNeasy plant mini kit (Valencia, CA). Reverse transcription experiments were performed using the SuperscriptIII first-strand synthesis kit (Invitrogen). qRT-PCR experiments were performed using 0.2 mM deoxynucleotide triphosphate, 0.2 mM primers and 1 unit of Taq polymerase (Banglo GENIE). PCR reactions were run for 25 or 28 cycles, which was determined in preliminary experiments to be in the linear range for these cDNA concentrations. Oligonucleotides for primers were purchased from IDT. The resulting cDNA was diluted with nuclease-free water to a final concentration of 2.5 ng/µL. All PCR reactions (10 mL) were assembled in triplicate and contained 5 mL of 23 Fast-SYBR Green Master mix (Applied Biosystems), 2.5 pmol of forward and reverse primer and 2 µL of template. “No RT” and “no template” control reactions were also included for each target gene. PCR reactions were cycled 10 min at 95°C followed by 40 cycles of 95°C for 15 sec and then 60°C for 1 min. A dissociation curve was added to the end of each assay to ensure that single products were amplified. Baseline and cycle thresholds were automatically assigned using Applied Biosystems 7500 Fast System software, and each amplification plot and dissociation curve was visually inspected for integrity. Cts values were exported into Microsoft Excel, and averages of triplicate reactions were determined.