Original research
A High-Throughput Method for Screening Arabidopsis Mutants with Disordered Abiotic Stress-Induced Calcium Signal

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Abstract

It is established that different stresses cause signal-specific changes in cellular Ca2+ level, which function as messengers in modulating diverse physiological processes. These calcium signals are important for stress adaptation. Though numbers of downstream components of calcium signal cascades have been identified, upstream events in calcium signal remain elusive, specifically components required for calcium signal generation due to the lack of high-throughput genetic assay. Here, we report the development of an easy and efficient method in a forward genetic screen for Ca2+ signals-deficient mutants in Arabidopsis thaliana. Using this method, 121 mutants with disordered NaCl- and H2O2-induced Ca2+ signals are isolated.

Introduction

Plants react to the changing environmental conditions through immediate signal transduction pathways. One integral part of many signal transduction pathways, in both plant and animal cells, is the usage of free calcium ions (Ca2+) as secondary messengers. Changes in cytosolic free calcium ([Ca2+]c) are apparent during the transduction of a very wide variety of abiotic signals including light, UV irradiation, low and high temperature, touch, hyperosmotic stress, salinity stress and oxidative stress (Knight et al., 1991, 1996, 1997; Frohnmeyer et al., 1999; Rentel and Knight, 2004). As abiotic stresses contribute to a great deal of agricultural loss, it is significant to understand how calcium signaling system works in plant under adverse environmental conditions.

In plants, most of the researches on Ca2+ signaling during abiotic stresses have been focused on the components involved in decoding of cytoplasmic Ca2+ signals (Xiong et al., 2002; Kudla et al., 2010). In the calcium signaling cascades of abiotic stress responses, many calcium-binding protein families functioned as sensor responders and sensor relays have been identified and proved to be important at the molecular level including CDPKs (Calcium-Dependent Protein Kinase), CaMs (Calmodulin) and SCaBPs/CBLs (Calcineurin B-like) (Haeseleer et al., 2002; Hrabak et al., 2003; Gong et al., 2004). In another aspect, the regulation of Ca2+ flux is a keystone of the integration of environmental signals and their translation into adaptive physiological responses. The generation of stimulus-specific Ca2+ oscillations depends on the spatial and temporal control of Ca2+ fluxes, which is involved with cell organelles, calcium channels, calcium pumps, stimuli sensors, other signal moleculars, and upstream regulators (Berridge et al., 2003). However, it is not well understood how [Ca2+]c changed and be regulated in plant. Not as animals, the clear identification of channels that are able to bind signaling intermediates that trigger Ca2+ release from internal stores, such as inositol-1,4,5-triphosphate (Gilroy et al., 1990), inositol hexakisphosphate (Lemtiri-Chlieh et al., 2003), cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, are absent (Leckie et al., 1998; Navazio et al., 2000). Ca2+-permeable channels play a role in shaping the Ca2+ signature by transporting Ca2+ into cells. Many classes of Ca2+ currents across the plasma membrane and some direct effectors of the Ca2+ influx have been identified by electrophysiological approaches but not genetically in plant. Several calcium channel proteins including MSCCs, CNGCs, GLRs and TPCs have been proved functional in mediating calcium signal generation in different stimuli (Jammes et al., 2011). But many functional channel genes as well as other [Ca2+]c shaping components are still waiting to be found.

Lacking an efficient method to isolate mutants in Ca2+ signal generation process may limit plant calcium signal study. Typical forward genetic analysis is always useful to find mutants and genes in specific pathway. Based on the availability of complete genome sequence, T-DNA insertion mutations can now be identified in any Arabidopsis gene using PCR-aided mutant screening approaches and large-scale sequencing of amplified insert junctions (Szabados et al., 2002). Here, we report the development of a new high-throughput method to find mutant disordered in calcium signature process that is easy to conduct and highly reproducible. Using this method, we carried out a forward genetic screen to look for deficient or excessive abiotic stress-induced Ca2+ influx, which led to the isolation of a few positive candidates.

Section snippets

Plant materials and mutants pool construction

Arabidopsis thaliana wild-type plant (Col-0) was transformed with 35S:apoaequorin construct pMAQ (Knight et al., 1991). The transformants expressing apoaequorin stably were named ca and ca was used to construct T-DNA insertion mutant pool. ca was then transformed with T-DNA vector pSKI105. Basta-resistant seedlings (T1 generation) were isolated and transferred to soil at a density of 10 seedlings per pot. T2 generation seeds were collected in bulk from each pot containing ten plants. These

NaCl and H2O2 induce an obvious Ca2+ concentration related luminescent signal in Arabidopsis seedlings constitutively expressing apoaequorin

The photoprotein aequorin is a calcium-sensitive luminescent protein. It enables changes in [Ca2+]c to be detected when the apoaequorin polypeptide and the luminophore coelenterazine bind three Ca2+ ions, which causes coelenterazine to be oxidized to coelenteramide, with the concomitant emission of blue light. This emitted blue light can be detected by a luminometer or can be imaged and the intensity of luminescence is depended on Ca2+ concentration. Knight et al. (1991) firstly applied this

The significance to isolated mutants in calcium signal generation pathway

Aequorin-base measurement of Ca2+ concentration was widely used in animal and plant calcium signaling studies. In Arabidopsis, this method was applied in measuring [Ca2+]c changes induced by touch, cold, oxidative stress, salt and drought (Knight et al., 1991, 1996, 1997; Rentel and Knight, 2004). Up to date, there is no report about using this method in a forward genetic study to screen mutants defective in cellular calcium changes. According the result report above, we introduced a

Acknowledgements

This work was supported by the National Funds for Distinguished Young Scientists in China (Grant No. 31025003) to Y. Guo.

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