научная статья по теме THE ABA-BINDING PROTEIN AA1 OF LUPINUS LUTEUS IS INVOLVED IN ABA-MEDIATED RESPONSES Биология

Текст научной статьи на тему «THE ABA-BINDING PROTEIN AA1 OF LUPINUS LUTEUS IS INVOLVED IN ABA-MEDIATED RESPONSES»

ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 2, с. 176-185

= ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

УДК 581.1

THE ABA-BINDING PROTEIN AA1 OF Lupinus luteus IS INVOLVED IN ABA-MEDIATED RESPONSES1

© 2015 A. V. Demidenko*, N. V. Kudryakova*, N. N. Karavaiko*, A. S. Kazakov**, G. N. Cherepneva*, G. V. Shevchenko*, S. E. Permyakov**, O. N. Kulaeva*, R. Oelmuller***, V. V. Kusnetsov*

* Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow ** Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino,

Moscow oblast

*** Institute of General Botany and Plant Physiology, University of Jena, Jena, Germany

Received August 27, 2014

We characterized AA1 (Abscisic acid Activated 1), a protein from Lupinus luteus L. predicted to be located in the apoplastic space who's mRNA and protein levels are strongly regulated by ABA, salt stress, and hypothermia. A fragment from the recombinant AA1 protein binds ABA as shown by the spectrofluorimetric titration assay of the protein by ABA. The BLAST software of the DFCI database identified more than 200 ESTs from 46 dicots and monocots, including three genes with unknown function from Arabidopsis thaliana, which are closely related to the lupine AA1. The central part of the proteins encoded by these genes contains the TolB motif from Escherichia coli and shares conserved WD40-like repeats, which form the basis for the tertiary beta-propeller structure and provide a potential platform for the assembly of protein complexes. Our data suggest that the highly conserved AA1 proteins from L. luteus and other higher plants are involved in ABA-mediated responses.

Keywords: Lupinus luteus - Arabidopsis thaliana - abiotic stress - ABA-binding protein - phytohormones -differential display --gene expression

DOI: 10.7868/S0015330315020050

INTRODUCTION

Phytohormones, in particular ABA, are important for adaptation mechanisms of plants to stressful environmental conditions. ABA is involved in the protection of plants against a wide range of environmental stressors, such as drought, salinity, cold, as well as pathogen attack. The hormone plays an important role in the colonization of ecological niches where water availability is limited or unstable [1]. Understanding the mechanisms of ABA perception and signaling also attracts attention for improving drought tolerance of cereals and other crops [2].

1 This text was submitted by authors in English.

Abbreviations: AA1 - Abscisic acid Activated 1; Ab - antibodies; BA - benzyladenine; DPP IV - IV dipeptidyl peptidases; FITC - fluorescein isothiocyanate; IgG - immunoglobulins; 154N-AA1 - coding sequence of AA1 gene in vector pQE-30 including the sequence encoding the 6xHis and additional 5 amino acids from the pQE-30 vector.

Corresponding author. Victor V. Kusnetsov. Botanicheskaya ul. 35, Moscow, 127276 Russia; Timiryazev Institute of Plant Physiology, Russian Academy of Sciences; fax. (+7) 499-977-8018; e-mail. vkusnetsov2001@mail.ru

An important area of ABA signaling is the identification of ABA-binding proteins or receptors. The existence of receptor sites for ABA binding was postulated about two decades ago [3], and many proteins with ABA-binding features were identified in the last decade [4]. The best characterized and most favored candidates for ABA receptors are members of the PYR/PYL/RCAR protein family. A variety of methods, including Rontgen structural analyses ofABA-re-ceptor complexes [5], mutants inactivated in PYR/PYL/RCAR genes and the analyses of a minimal ABA signal transduction pathway in protoplasts [6] demonstrated that members of the PYR/PYL/RCAR family are ABA receptors characterized by specific hormone recognition and signal transduction [7].

However, at least 10 more potential ABA receptors have been proposed. ABAP1 from barley seed aleu-rone, the receptor-like protein kinases RPK1 from Arabidopsis thaliana, ABAR/GUN5/CHLH identified as H subunit of the heterotrimeric Mg+-chelatase complex from A. thaliana and its homolog from Vicia faba, which have been characterized as ABA-binding

membrane proteins, the putative GPCR-protein GCR2 and its homologues GCL1 and GCL2 [4], as well as the GTG1 and GTG2 receptors bound to het-erotrimeric G-proteins [8].

The description of other ABA-binding proteins, the complexity of ABA signaling [9], and the integration of ABA in numerous signaling networks, which include second messengers such as Ca2+, phosphorylation cascades, phosphoinositides, phosphatidic acid, and reactive oxygen species [1, 10] suggest that additional components involved in ABA perception and signaling may exist.

We identified a novel gene (Abscisic acid Activated 1, AA1) from Lupinus luteus L., which was activated by ABA and inhibited by cytokinin. The objective of this work was to study the physicochemical properties and biological role of AA1 from L. luteus.

MATERIALS AND METHODS

Growth of yellow lupine seedlings. Seeds of yellow lupine (Lupinus luteus L., cv. Akademicheskii I) were sterilized with concentrated sulfuric acid for 10 min, scarified, and germinated in the climatic chamber at 23°C in darkness for 3 days on moist tissue paper. The cotyledons were cut in dim green light and kept in darkness for further 24 h on water to decrease endogenous cytokinin and ABA levels. They were then placed in Petri dishes on tissue paper soaked with water or solutions containing either ABA (7.6 x 10-5 M) or cytokinin (BA, 2.2 x 10-5 M) in darkness or high-intensity white light (120 ^mol/(m2 s)). To study the effects of abiotic stressors, lupine was grown in trays on moist tissue paper in darkness until the 9th day; then seedlings were transferred to 150 mM NaCl solution (the concentration was optimized in pilot experiments) or cooled to 4°C. The plant material was fixed in liquid nitrogen 1, 2 or 3 days after the start of the experiment.

DNA isolation and Southern analysis. Genomic DNA was isolated from lupine cotyledons as described by Sambrook et al. [11]. The DNA was digested with the indicated restriction enzymes, and DNA fragments were separated on a 1% agarose gel (10 ^g DNA per line) before transfer to nylon membranes. Filters were hybridized to radiolabeled DNA fragments [11].

RNA isolation and northern analysis. Total RNA was extracted from lupine cotyledons using TRiozol reagent ("Gibco/BRL", United States) according to the manufacturer's protocol. RNA was electrophore-sed on a 1.2% agarose-formaldehyde gel and blotted onto Hybond-N+ membranes ("Amersham Pharmacia Biotech", England) by capillary transfer [11].

Radioactive probes for hybridization were produced by PCR in the presence of [a-32P]-dCTP. PCR-generated and purified fragments of the corresponding genes served as templates. RNA gel-blot hybridization with [32P]-labeled probes and subsequent

membrane washing were carried out as described [11]. Radioactive signals were detected and quantified using a Phosphorimager (Typhoon Trio+, "GE Healthcare", United States) or by autoradiography.

Differential display. The differential display method was mainly performed according to Liang et al. [12], as specified in the protocol of the GenHunter mRNA Differential Display Kit ("GenHunter Corporation", United Kingdom). The differential display method was performed as described in the Supplementary Method 1.

Bioinformatic methods used in the work are described in the Supplementary Method 2.

AA1 constructs. A full-length cDNA for AA1 (2137 bp) from the phage library (^gt11) of yellow lupine was cloned into the Eco RI site of the pBlueScript II KS+ ("Stratagene") vector. A BamHI restriction site was created by the replacement of A241 of the cDNA by a C. After amplification, the modified cDNA fragment was restricted with BamHI and HindIII and cloned into the same sites of the pQE-30 vector. Thereafter, the 3' -region of the cloned sequence was removed by restriction at the ClaI site, an internal restriction site in AA1, and HindIII. As a result, a 429 bp-long AA1 fragment remained in the pQE-30 vector; it corresponds to the region 236 to 664 bp of the initial cDNA. The size of coding sequence of 154N-AA1 in vector pQE-30 is 462 bp (including the sequence encoding the 6xHis and additional 5 amino acids from the pQE-30 vector).

Overexpression and purification of the recombinant protein using affinity chromatography on Ni-NTA Sepharose. The cDNA fragment (429 bp of AA1 plus additions) was expressed in the M15 strain of E. coli. The expressed protein represented the fragment of AA1 comprising 143 amino acid residues, the 6xHis tag, and additional 5 amino acids from the pQE-30 vector (mol wt of expressed protein was 17.5 kD). The produced recombinant protein was purified by affinity chromatography on the Gravity Flow column packed with Ni-NTA-Sepharose ("Qiagen") as recommended by the manufacturer.

Production of antibodies (Ab) against the recombinant protein. The recombinant protein purified on Ni-NTA-Sepharose was used for rabbit immunization to obtain polyclonal antibodies (Ab) [13]. To increase primary Ab specificity to the AA1 fragment, the serum was exhausted on a membrane coated with the recombinant protein. The IgG fraction was purified by affinity chromatography on ProteinG-Sepharose ("Sig-maAldrich") as recommended by the manufacturer. The IgG preparations were mixed with 50% glycerol, frozen in liquid nitrogen, and stored at -70°C.

Protein extraction and western analysis. Protein extraction from lupine was performed as described by Conlon and Salter [14] with some modifications. The protein concentration was determined by the bicin-choninic acid assay [15]. Immunoblotting was per-

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Fig. 1. Southern analysis of AA1.

DNA from lupine cotyledons was digested with HindIII (I), Xbal (II), or £coRI (III). The DNA fragments were separated on an agarose gel, transferred on the membrane, and hybridized to the radiolabeled AA1 probe. Numbers show DNA sizes in bp.

formed as described in [16]. The proteins were tran

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