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Plant Physiology and Biochemistry 58 (2012) 1e5

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Plant Physiology and Biochemistry

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Short communication

Wide range of interacting partners of pea Gb subunit of G-proteins suggestsits multiple functions in cell signalling

Deepak Bhardwaj a,b, Suman Lakhanpaul a, Narendra Tuteja b,*

aDepartment of Botany, University of Delhi, Indiab International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

a r t i c l e i n f o

Article history:Received 26 April 2012Accepted 5 June 2012Available online 19 June 2012

Keywords:Climate changeG-proteinsG-beta subunitPisum sativumYeast-two-hybridInteracting partnersSignal transduction

* Corresponding author. Tel.: þ91 11 26742357; faxE-mail address: narendra@icgeb.res.in (N. Tuteja).

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.06.005

a b s t r a c t

Climate change is a major concern especially in view of the increasing global population and foodsecurity. Plant scientists need to look for genetic tools whose appropriate usage can contribute tosustainable food availability. G-proteins have been identified as some of the potential genetic tools thatcould be useful for protecting plants from various stresses. Heterotrimeric G-proteins consisting of threesubunits Ga, Gb and Gg are important components of a number of signalling pathways. Their structureand functions are already well studied in animals but their potential in plants is now gaining attentionfor their role in stress tolerance. Earlier we have reported that over expressing pea Gb conferred heattolerance in tobacco plants. Here we report the interacting partners (proteins) of Gb subunit of Pisumsativum and their putative role in stress and development. Out of 90 transformants isolated from theyeast-two-hybrid (Y2H) screening, seven were chosen for further investigation due to their recurrence inmultiple experiments. These interacting partners were confirmed using b-galactosidase colony filter liftand ONPG (O-nitrophenyl-b-D-galactopyranoside) assays. These partners include thioredoxin H,histidine-containing phosphotransfer protein 5-like, pathogenesis-related protein, glucan endo-beta-1,3-glucosidase (acidic isoform), glycine rich RNA binding protein, cold and drought-regulated protein(corA gene) and soluble inorganic pyrophosphatase 1. This study suggests the role of pea Gb subunit instress signal transduction and development pathways owing to its capability to interact with a widerange of proteins of multiple functions.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

G-proteins are important signalling molecules in plants. Theyconsist of three subunits Ga, Gb and Gg and the number of eachsubunit in various plants can vary [1]. G-proteins remain attachedto G-protein coupled receptor (GCR1) or can act through regulatorof G-protein signalling (RGS) and with the reception of the signalfrom elicitors the heterotrimer disassociates rendering the subunitsto act independently and thus allowing Ga and Gbg interactionswith other cellular proteins [2e4]. With the recent discovery ofArabidopsis G-protein intractome, it is now evident that plant G-proteins are multifunctional and play a significant role in thedevelopment and environmental stresses which further need to beexplored in the wake of global climate change [5].

The Gb subunit, which contains WD40 domains is reported tobe involved in many significant functions and processes of the

: þ91 11 26742316.

son SAS. All rights reserved.

plants such as development of leaves, gynoecium, stomata, androot and also in pathogen resistance and alleviating heat stress[6e11]. RGB1 mutants of rice Gb are shorter in height and havesmall sterile seeds [12].

Recently, it has been shown that Arabidopsis has one more Ggsubunit called Gg3 apart from Gg1 and Gg2; also these three Gg’sare equivalent to Gb subunit as the triple Gg1Gg2Gg3 mutant hasan identical phenotype to the agb1 mutant [13,14]. PsGb subunitalso has role in heat tolerancewhich has been earlier proved in overexpressing lines of tobacco-transformed with 35S-PsGb [11].Moreover, AGB1 is also involved in the resistance againstnecrotrophic fungi [9]. Though, the effect of Gb subunit is versatileand well known but the intricate pathway involved in heat toler-ance and resistance against pathogens is yet to be deciphered. Inorder to address the question related to elucidation of the pathwayinvolved in these processes, we used Pisum sativum Gb subunit(PsGb) and identified its interacting proteins (IPs). Here, we reportthe interacting partners of PsGb cloned from the Y2H studies. From90 IPs that we got initially, seven IPs were chosen for furtherinvestigation due to their recurrence in multiple experiments.

Fig. 1. Yeast-two-hybrid system-based interaction between PsGb and stress inducedcDNA library cloned in pGADT7 vector. AeB: Colonies grown on a synthetic dextroseplate lacking tryptophan and leucine (2-Drop out; 2DO); CeD: Colonies grown onsynthetic dextrose plate lacking tryptophan, leucine and histidine(3-Drop out; 3DO);EeF: Colonies grown on a synthetic dextrose plate lacking leucine, tryptophan andhistidine having 5 and 30 mM 3-AT(3-Amino-1, 2, 4-triazole); GeH: Same colonieswere analysed for the b-galactosidase filter lift assay.Colonies 2,10,17, 32,35,36 and 39are the clones that grew on the 30 mM 3-AT, whereas V(vector control); S(straincontrol) 18, 19, 33,34 and 38 were used as negative control.

D. Bhardwaj et al. / Plant Physiology and Biochemistry 58 (2012) 1e52

Selected interacting partners were confirmed using colony filter liftand ONPG assay.

2. Results and discussion

G-proteins are widely known to play a significant role in variousbiological processes in living organisms. However, the precisemechanism and their role in plants are far from understood. Inorder to isolate the interacting partners (IPs) of Gb in pea, weperformed Gal4 based yeast-two-hybrid (Y2H) screening assay.P. sativum cDNA library cloned in yeast pGADT7 vector was used asa prey and P. sativum Gb cloned in pGBKT7 was used as bait. Boththe gene and library were co-transformed into yeast strain AH109and a total of about 90 positive colonies were observed in two-dropout (2 DO)-SD medium (Fig. 1A). These 90 colonies were randomlyselected and were shifted to 3 DO-SD (Fig. 1B). Out of 90, fewcolonies that showed healthy growth even after 15 days werepatched on 3 DO-SDþ5 mM 3-AT [3-Amino- 1, 2, 4-triazole](Fig. 1C). Seven colonies that showed healthy growth for 15 daysexhibited similar growth pattern when patched on higherconcentration of 3 DO-SDþ30 mM 3-AT, thus confirming stronginteraction. These experiments were repeated thrice to avoid anypossibility of false negative (Fig. 1D). Subsequently, the samecolonies were tested for b-galactosidase activity using colony filterlift assay and all of them showed the expected blue colour (Fig. 1E).No significant colony growth at this selection medium wasdetectable in negative control experiments, where the AH109 wastransformed with empty vectors. The ONPG (O-nitrophenyl-b-D-galactopyranoside) assay was also performed for all the sevencolonies for measuring strength of interaction following high-throughput quantitative b-galactosidase assay [15]. Significant b-gal activity was found in all the seven but colonies 35 and 39showed higher activity then the other five (Fig. 2). No b-gal activitywas observed in the negative controls co-expressing empty vectors.Interestingly, all IPs were found common in the three repetitiveexperiments. Sequences of these seven clones were analysed forhomology and the accession numbers of the genes showing highsimilarity with them along with their putative functions are givenin Table 1. These genes have been reported to play significant role inenvironmental stresses and plant development (Fig. 3). In fact, twoout of seven interacting partners, namely, pathogenesis-relatedprotein and glycine rich protein have been reported in Arabi-dopsis interactome [5]. All the experiments from Y2H to b-Gal(Colony filter lift assay and ONPG) were repeated three times andsame interacting partners were found in these three replications.

Role of Gb subunit of G-proteins has been shown in manycellular and physiological responses of plants. Our results suggestthe involvement of Gb in defence and other signals with regard tostress related pathway. Two P. sativum thioredoxin proteinsPsTRXh1 and PsTRXh2 have been reported to be involved in redox-regulation by interacting with different targets proteins [16].Interestingly, it has already been shown that one of target of thio-redoxin during seed germination in case of model legumeMedicagotruncatula was Gb subunit along with other proteins such as UDP-Glc pyrophosphorylase and glycine rich RNA binding protein [17].Other IP namely, histidine-containing phosphotransfer protein isan important component of cytokinin signalling pathway. Stimu-lation of GCR1 in response to cytokinin has been suggested earlierbut was not pursued further due to the lack of evidence [18].Cytokinin signalling is mediated by a HIS-ASP phosphorelaypathway. This pathway consists of a two-component system withhistidine kinase as receptor for perceiving signal and a regulatorwhich has conserved receiver domain that gets phosphorylated byhistidine kinase [19]. Since, cytokinin is known to be associatedwithmany developmental and phenotypic effects which are similar

to the functions of G-proteins such as cell division, root develop-ment, shoot growth and senescence. The cross talk between G-protein and cytokinin mediated pathway through the involvementof histidine-containing phosphotransfer protein is herebyproposed. Similarly, defensin and glucan endo-beta-1, 3-glucosidase (acidic isoform) belong to the family of pathogenesis-related protein (PR proteins) which are involved in wide range ofphysiological and developmental signalling processes [20]. Despitethe presence of defensin genes in case of agb1 mutants, their

Fig. 2. Comparative analysis of interacting partners of PsGb protein. Seven interacting partners that were found in the multiple experiments grew better on SD lacking LTHþ30 mM3AT and were subjected to ONPG (O-nitrophenyl-b-D-galactopyranoside) assay for measuring interaction strength.

D. Bhardwaj et al. / Plant Physiology and Biochemistry 58 (2012) 1e5 3

susceptibility to pathogens has been reported [9]. Hence, interplaybetween Gb and these defence related protein may be important ingiving resistance to the plant against pathogens. Glycine rich RNAbinding protein (GRRBPs) as well as cold and drought-regulatedproteins were also found to be interacting with PsGb in thepresent study. Interestingly, membrane located beta subunit hasbeen found in the nuclei purified from the Tobacco leaves [21,22].Though the exact mechanism and function of Gb entering nucleusis not clear, an interaction between GRRBP and Gb could be thepossible clue for this phenomenon. An interaction of soluble inor-ganic pyrophosphastase with PsGb is hereby reported for the firsttime. Inorganic pyrophosphastase is an electrogenic pump thatcatalyses reactions related to the hydrolysis of pyrophosphate forthe generation of energy to transport protons from cytosol tovacuole and is also found in the plastids [23]. Though an interactionof Gb with an electrogenic pump has not been reported, the regu-lation of such channels through proteineprotein interaction islikely due to their proximity to the cell membranes.

3. Conclusions

This study suggests the role of pea Gb subunit in developmentand stress signalling owing to its capability to interact with a widerange of proteins of multiple functions highlighting the importanceof G-proteins in plants. It underscores a need to further decipherthe pathways and their components that enable the plants tocombat various environmental challenges. Genetic manipulation ofPsGb and its interacting partners is an important research that willcontribute towards developing stress resistant crop cultivars for thebetter survival in the context of climate change. The findings pre-sented here will help foster a better understanding of our knowl-edge regarding G-proteins signalling in plants under stressconditions.

Table 1List of the selected interacting partners of PsGb (bait) identified and their putative funct

S.No. Interacting partner (prey)

1 Pisum sativum mRNA for thioredoxin h (trxh gene)2 Glycine max histidine-containing phosphotransfer protein 5-like mRNA3 P. sativum pathogenesis-related protein mRNA, complete cds4 Cicer arietinum mRNA for glucan endo-beta-1, 3-glucosidase, acidic isofo5 Pisum sativum Glycine rich RNA binding protein6 Cold and drought-regulated protein; corA gene (putative glycine rich pro7 Medicago truncatula clone MTYF9_FA_FB_FC1G-H-3 soluble inorganic py

4. Material and methods

4.1. Plant growth and stress treatments

Pea (P. sativum) seeds were germinated in sterilized wetvermiculite under a 14 h/10 h light/dark cycle at 25 �C for 7 days.After 7 days, plants were given several treatments like MeJA, ABA,H2O2 and heat stress for 12 h; leaves were frozen in liquid nitrogenjust before RNA was isolated using RNeasy Plant Mini Kit fromQiagen (catalogue no.74904) following the manufacturer’sinstructions. Finally, these RNAs were used for synthesizing thecDNAs. All the cDNAs were mixed together and the yeast AD librarywas made out of it.

4.2. Construction of P. sativum cDNA library and cloning of PsGbfrom pea

A cDNA library was constructed from 5 mg of poly(A) RNA (iso-lated 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 unitsper ml. For cloning of the Gb subunit, the pea cDNA library wasscreened using a radiolabeled tobacco Gb subunit as probe asdescribed earlier [7]. cDNA sequencing was done by Macrogen,Korea (http://www.macrogen.com/eng/sequencing/dua.jsp). Ahomology search was performed using BLAST (NCBI), (http://www.ncbi.nlm.nih.gov/BLAST).

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

A Gal4 based two-hybrid system was used as described by themanufacturer (Clontech, USA; http://www.clontech.com/). The

ions.

Accession number Putative function(s)

AJ310990.1 Heat and oxidative stressXM003518890 Cytokinin signalingFJ174689.1 Biotic stress

rm AJ012751.1 Biotic stressU81287 Cold stress and splicing

tein precursor) AJ831469.1 Cold and drought stressrophosphatase 1 BT051944 Heat, drought and salt stress

Fig. 3. Hypothetical model of action of G-proteins. The G-proteins are elicited by various abiotic and biotic elicitors like ABA, methyl jasmonate, brassinosteroids and pathogens.These elicitors sensitize the putative receptors such as GCR1 and RGS causing splitting of G-protein subunits (Ga and Gbg). Ga and Gbg act as signalling molecules by interactingwith other cellular proteins. PsGb interacts with thioredoxin H, histidine-containing phosphotransfer protein 5-like, pathogenesis-related protein, glucan endo-beta-1, 3-glucosidase (acidic isoform), glycine rich RNA binding protein, cold and drought-regulated protein (corA gene) and soluble inorganic pyrophosphatase 1.

D. Bhardwaj et al. / Plant Physiology and Biochemistry 58 (2012) 1e54

coding region of the Gb subunit (1134 bp) was amplified by PCRwith primers harbouring restriction sites and cloned in-frame intothe EcoRI and BglII sites of the binding domain vector pGBKT7. Theresulting vector pGBKT7-PsGb was co-transformed with P. sativumstress induced AD library into yeast strain AH109 harbouring tworeporter genes (HIS3 and b-galactosidase) by the lithium acetatemethod. AH109 contains integrated copies of ADE2, HIS3 and lacZ(MAL1) reporter genes under the control of distinct GAL4 upstreamactivating sequences TATA box and UAS sequences. Thesepromoters yield strong and very specific responses to GAL4. Yeastcells carrying both the plasmids were selected on the syntheticmedium lacking Leu and Trp (SD-Leu-Trp-). The yeast cells werethen streaked onto a SD medium ((Leu), (Trp), (His)) containing30 mM 3-AT (3-Amino-1, 2, 4-triazole) to determine expression ofthe HIS3 nutritional reporter. The b-galactosidase expression of theHisþ colonies was analysed by colony filter lift and ONPG assays asdescribed by the manufacturer (Clontech). Seven interactingproteins were identified by homology search using BLAST (NCBI),(http://www.ncbi.nlm.nih.gov/BLAST)

Acknowledgements

Authors acknowledge the University Grant Commission andDepartment of Science & Technology (DST), New Delhi, India forfunding this research.

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