Tài liệu Báo cáo khoa học: Cloning and characterization of CBL-CIPK signalling components from a

Cloning and characterization of CBL-CIPK signalling

components from a legume (Pisum sativum)

Shilpi Mahajan, Sudhir K. Sopory and Narendra Tuteja

Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India

In plants, calcium plays an important role in regula￾ting gene expression and many other processes inclu￾ding abiotic stress signalling. However, the molecular

mechanisms underlying the role of calcium in cellular

functions are not well established. Many external

stimuli including light and various stress factors can

bring out changes in cellular Ca2+ level, which can

affect plant growth and development [1,2]. The Ca2+

serves as second messenger and its concentration is

delicately balanced by the presence of ‘Ca2+ stores’

such as vacuoles, endoplasmic reticulum, mitochon￾dria and cell wall. Ca2+ signals exhibit a high degree

of specificity and are decoded by Ca2+ sensing

proteins known as Ca2+ sensors, which are small

proteins interacting with their target proteins to relay

the signal. In plant cells many Ca2+ sensors have

been identified which include calmodulin (CaM) and

calmodulin-related proteins [3,4], Ca2+-dependent

protein kinases (CDPKs) [5,6], and calcineurin B-like

proteins (CBLs) [4]. The first plant CBL to be iden￾tified was from Arabidopsis thaliana, and is known

as both AtCBL and ScaBP (SOS3-like calcium￾binding protein) [7,8]. CBL proteins contain four

Ca2+-binding EF hand motifs [9] and functions by

interacting and regulating a group of Ser⁄Thr pro￾tein kinases called CBL-interacting protein kinases

Keywords

abscisic acid; abiotic stress; biotic stress;

calcium sensor CBL; CIPK

Correspondence

N. Tuteja, Plant Molecular Biology,

International Centre for Genetic Engineering

and Biotechnology, Aruna Asaf Ali Marg,

New Delhi, 110067, India

Fax: +91 11 26162316

E-mail: [email protected]

Note

The sequences reported in this paper have

been deposited in the General Bank

database (accession nos. AY134619 (pea

CBL cDNA); AY883569 (pea CBL genomic

clone); AY191840 (pea CIPK cDNA).

(Received 7 October 2005, revised 11

December 2005, accepted 19 December

2005)

doi:10.1111/j.1742-4658.2006.05111.x

The studies on calcium sensor calcineurin B-like protein (CBL) and CBL

interacting protein kinases (CIPK) are limited to Arabidopsis and rice and

their functional role is only beginning to emerge. Here, we present cloning

and characterization of a protein kinase (PsCIPK) from a legume, pea,

with novel properties. The PsCIPK gene is intronless and encodes a protein

that showed partial homology to the members of CIPK family. The recom￾binant PsCIPK protein was autophosphorylated at Thr residue(s). Immu￾noprecipitation and yeast two-hybrid analysis showed direct interaction of

PsCIPK with PsCBL, whose cDNA and genomic DNA were also cloned in

this study. PsCBL showed homology to AtCBL3 and contained calcium￾binding activity. We demonstrate for the first time that PsCBL is phos￾phorylated at its Thr residue(s) by PsCIPK. Immunofluorescence ⁄ confocal

microscopy showed that PsCBL is exclusively localized in the cytosol,

whereas PsCIPK is localized in the cytosol and the outer membrane. The

exposure of plants to NaCl, cold and wounding co-ordinately upregulated

the expression of PsCBL and PsCIPK genes. The transcript levels of both

genes were also coordinately stimulated in response to calcium and salicylic

acid. However, drought and abscisic acid had no effect on the expression

of these genes. These studies show the ubiquitous presence of CBL⁄ CIPK

in higher plants and enhance our understanding of their role in abiotic and

biotic stress signalling.

Abbreviations

3-AT, 3-aminotrizole; ABA, abscisic acid; CaM, calmodulin; CBL, calcineurin B-like protein; CDPK, Ca2+-dependent protein kinase; CIPK, CBL

interacting protein kinases; DAPI, diamidino-2phenylindole hydrochloride; DTT, dithiothreitol; IPTG, isopropyl thio-b-D-galactoside; SA, salicylic

acid; SD, synthetic dextrose; UAS, upstream activating sequences; UTR, 5¢ untranslated region; YPD, yeast extract–peptone–dextrose.

FEBS Journal 273 (2006) 907–925 ª 2006 The Authors Journal compilation ª 2006 FEBS 907