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Plant Physiology 132:728-731 (2003) © 2003 American Society of Plant Biologists A Cyclic Nucleotide-Gated Ion Channel, CNGC2, Is Crucial for Plant Development and Adaptation to Calcium Stress1Biotechnology Center (C.W.M.C., L.M.S., M.R.S.) and Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin 53706 (R.K.S., A.F.B.)
To better understand the relationship between functions of cyclic nucleotide-gated channels (CNGCs) and plant physiology, we studied the effect of different ions on the growth of Arabidopsis cngc2 mutants. Here, we report the novel finding that cngc2 plants are specifically hypersensitive to calcium in their growth environments, leading to severe reductions in size and reproductive ability.
CNGCs are important for sensory transduction in animals. The best characterized CNGCs are from the visual and olfactory systems. This class of channels does not discriminate well between alkaline metals and allow a substantial Ca2+ flow. Physiologically relevant permeant ions are Na+, K+, and Ca2+, and in both visual and olfactory transduction, this Ca2+ influx is important for desensitization and adaptation (Zagotta and Siegelbaum, 1996
Little is known about the physiological functions of CNGCs in plants. However, recent works have suggested that at least some members are involved in uptake and homeostasis of heavy metals such as Ni2+ and Pb2+ (Arazi et al., 1999
In heterologous expression systems, CNGC2 can form an ion channel that mediates Ca2+ and K+ influxes but interestingly does not allow substantial Na+ influx (Leng et al., 1999
CNGC2 Mutant Alleles
Experiments reported herein utilized two homozygous mutant lines of Arabidopsis. The first allele is cngc2-1 and contains a premature stop codon at W290 (Clough et al., 2000
Both cngc2-1 and cngc2-2 plants showed Ca2+-hypersensitive growth phenotypes. Growth of mutant plants on Murashige and Skoog control media (with or without 1% [w/v] supplemented Suc) was not significantly different from their respective wild types for about the first 3 weeks under our normal growth condition (for details, see "Materials and Methods"). However, when the media contained increased amounts of CaCl2, both mutants showed drastically stunted growth. A representative example is shown in Figure 1. This phenotype was clearly visible after about 12 d of growth, and mutants remained smaller than their wild-type counterparts by all measurable criteria (rosette size, root mass, and overall stature) throughout the rest of their life spans. Whereas wild-type plants were not affected by moderate increases in [Ca2+] (up to 30 mM extra CaCl2 and growth for up to 6 weeks on petri plates), both mutants showed significant size reduction in all [Ca2+] tested (Fig. 2A; Student's t test, P « 0.001). This growth defect was specific to Ca2+, because mutants and wild types were indistinguishable in their responses to changes in various other ions. Conditions tested include: increasing [Mg2+] (up to 20 mM additional MgCl2), [K+] (up to 100 mM additional KCl), and [Na+] (up to 75 mM additional NaCl), and a 10-fold increase or decrease in [H+] (Student's t test showed no significant difference, P < 0.05; also, see below). As an example, Figures 1 and 2B show that the growth of mutants and wild types on increasing [NaCl] was very comparable. Taken together, these results suggest that cngc2 mutants are not hypersensitive to ionic or osmotic stresses in general but rather are specifically impaired in coping with excess external Ca2+. cngc2 mutants maintained their Ca2+ sensitivity in later stages of development. We can grow plants in magenta cubes on Murashige and Skoog media to maturity. After about 2 months, wild-type plants had healthy seed sets despite the addition of Ca2+ to the medium (10–30 mM). Conversely, cngc2 mutants in such conditions were severely dwarfed and produced no or very few seeds (data not shown). This shows that defects in cngc2 that are exaggerated in elevated external [Ca2+] affect both vegetative and reproductive development.
When grown directly in a soil mixture, mutant plants similarly showed hypersensitivity to Ca2+. Analogous to the scenario described above, the size difference between soil-grown mutant and wild-type plants was exacerbated when they were watered with 10 mM CaCl2 instead of distilled water (Fig. 3). Whereas wild-type plants were minimally affected by watering with 10 mM CaCl2 in both size and seed sets, mutants were significantly stunted in vegetative and reproductive growth and led to a complete absence of seed production (Fig. 3; data not shown).
We show that null mutations in CNGC2 lead to a specific and dramatic Ca2+ hypersensitivity that results in severe reductions in plant size and seed yield. To our knowledge, this is the first report of a significant and stable phenotype caused by a single gene mutation when plants are challenged with a nominal increase in extracellular [Ca2+]. The only other mutant we are aware of with Ca2+-sensitive growth is smt1. Such plants are defective in a sterol methyl-transferase, leading to Ca2+ sensitivity in roots on agar media during the first 2 weeks of growth (Diener et al., 2000  ). This is in stark contrast to cngc2 mutants, which display diminutive growth through different developmental stages and growth media. Our finding is especially intriguing because the Arabidopsis genome potentially contains 19 other CNGCs (see http://www.cbs.wmn.edu/Arabidopsis/), and yet disrupting the function of CNGC2 alone has such a large impact. Ca2+ levels used in this study are commonly found in natural growth environments (for example, see Kelling and Schulte, 1998). Therefore, our data support the hypothesis that CNGC2 is particularly important for plant growth in physiological calcified conditions.
We emphasize that cngc2 mutants are not hypersensitive to stress in general. The responses of cngc2 and wild-type plants to various other challenges are indistinguishable. These include increasing [Na+] and [Mg2+], increasing and decreasing pH and [K+], decreasing Ca2+ (by omitting Ca2+ from the medium or by chelating Ca2+ with 1.5 mM BAPTA), and cold and freezing conditions (see "Results"; C.W.M. Chan and M.R. Sussman, unpublished data). The phenotypic difference between mutant and wild-type plants upon increasing external [Ca2+] is not affected by the inclusion of 1% (w/v) Suc and is independent of the light regimen (we obtained qualitatively similar results with 8-h-light/16-h-dark cycles; data not shown). We also noticed that cngc2-2, just like cngc2-1, did not display the HR upon pathogen infection (G.I. Jurkowski and A.F. Bent, unpublished data; Yu et al., 1998
Mutant phenotypes cannot be attributed solely to the constitutive induction of salicylic acid and defense-related genes in cngc2. Depletion of salicylate compounds in nahG+ cngc2-1 transgenic plants removes the constitutively elevated pathogen resistance without completely reversing the dwarf and lack of HR phenotypes (Clough et al., 2000 One of the simplest explanations for the Ca2+-hypersensitive phenotype in cngc2 mutants is that mutant plants are dwarf due to Ca2+ toxicity. This Ca2+ toxicity can be the result of Ca2+ hyper-accumulation or of a defect in sensing and/or adapting to elevated levels of Ca2+ in the growth environment. Preliminary results suggested that mutant plants did not accumulate more Ca2+ than wild type either in control growth conditions or when placed under Ca2+ stress (C.W.M. Chan, J.F. Harper, and M.R. Sussman, unpublished data). Therefore, our current working model is that mutants are perhaps defective in signaling pathways that allow for normal growth in low tens of millimolar Ca2+.
In summary, our data support the hypothesis that CNGC2 is critical for plant development. Mutants are affected in vegetative and reproductive growth, programmed cell death, and adaptive responses to biotic and abiotic stimuli. Our data represent a starting point for further analyses into relationships between CNGC2, calcium signaling, and the aforementioned physiological processes. Importantly, our result strongly suggests that CNGC2 is a key determinant for growth under physiologically relevant [Ca2+].
Mutant Isolation
Two mutant Arabidopsis lines were used. cngc2-2 (WS ecotype) was isolated from the Wisconsin T-DNA
Plants were routinely grown on 0.5x Murashige and Skoog salt (Invitrogen, Carlsbad, CA), 2.5 mM MES, and 0.8% (w/v) washed agar (Sigma-Aldrich, St. Louis), with pH adjusted to 5.7 with KOH as necessary. Murashige and Skoog (0.5x) media contains 1.5 mM CaCl2. Media were supplemented with 1% (w/v) Suc and additional ions as indicated in the text. Alternatively, plants were grown in a soil mixture (Jiffy mix:medium grade vermiculite, 2:1 [v/v]) and watered with distilled water (or distilled water supplemented with 10 mM CaCl2 where indicated) about once a week. All plants were grown in growth chambers (AR-75L, Percival, Boone, IA) that were set to 21°C and constant light (about 40 µE). Received December 16, 2002; returned for revision December 21, 2002; accepted January 20, 2003.
www.plantphysiol.org/cgi/doi/10.1104/pp.102.019216.
1 This work was supported by the National Science Foundation (grant no. IBN–9728563 to M.R.S.) and by the U.S. Department of Agriculture (grant no. 95–37304–2364 to M.R.S.).
2 Present address: The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850. * Corresponding author; e-mail msussman{at}facstaff.wisc.edu; fax 608–262–6748.
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RESULTS
DISCUSSION
, Wassilewskija (WS); 
, cngc2-2; 
, Columbia (Col-0); 
, cngc2-1.B,Na+ response of cngc2 mutants. Fresh weights of plants grown on control media or media containing increasing amounts of NaCl were measured. Plants were grown on indicated media for 2 weeks, and the weight of aerial portions was measured from three groups of plants, each of 15 individuals. The average weight of an individual in each group was obtained, and then the average weights and corresponding SDs of individual plants in each growth condition were calculated and plotted. The result shows that mutant and wild-type plants are similarly sensitive to external NaCl. 
collection using standard methods (Krysan et al., 1999
