Salt-dependent regulation of a CNG channel subfamily in Arabidopsis

Abstract

Background: In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies.

Results: GUS, GFP and luciferase reporter assays were used to study the expression of CNGC19 and CNGC20 genes from Arabidopsis thaliana in response to developmental cues and salt stress. CNGC19 and CNGC20 were differentially expressed in roots and shoots. The CNGC19 gene was predominantly active in roots already at early growth stages. Major expression was observed in the phloem. CNGC20 showed highest promoter activity in mesophyll cells surrounding the veins. Its expression increased during development and was maximal in mature and senescent leaves. Both genes were upregulated in the shoot in response to elevated NaCl but not mannitol concentrations. While in the root, CNGC19 did not respond to changes in the salt concentration, in the shoot it was strongly upregulated in the observed time frame (6-72 hours). Salt-induction of CNGC20 was also observed in the shoot, starting already one hour after stress treatment. It occurred with similar kinetics, irrespective of whether NaCl was applied to roots of intact plants or to the petiole of detached leaves. No differences in K and Na contents of the shoots were measured in homozygous T-DNA insertion lines for CNGC19 and CNGC20, respectively, which developed a growth phenotype in the presence of up to 75 mM NaCl similar to that of the wild type.

Conclusion: Together, the results strongly suggest that both channels are involved in the salinity response of different cell types in the shoot. Upon salinity both genes are upregulated within hours. CNGC19 and CNGC20 could assist the plant to cope with toxic effects caused by salt stress, probably by contributing to a re-allocation of sodium within the plant.

Figure 1

CNGC19 expression in vascular tissues. GUS staining of plants at different ages carrying the CNGC19p:GUS construct: 2 (A), 4 (B), 18 (C), 39 (D, E) days after stratification. (F-H) Pictures of developing lateral roots where blue staining is not detected before vasculature formation. (I-K) GUS staining visible in the phloem strands of the root. (L) Cross section of the root with GUS staining in the phloem but not in xylem cells of the stele. The bar represents 20 μm. (M) Part of an adult leaf showing GUS staining in the vasculature. Note, that the plant shown in (M) has been grown on salt-containing agar (see Fig. 3).

Figure 2

CNGC20 expression in roots and shoots. GUS staining of plants carrying the CNGC20p:GUS construct was observed in roots of young seedlings (A), in petioles and tissue surrounding the veins of adult leaves (B), and carpels (C). On the cellular level, GUS staining was detected in the root cortex (D), mesophyll surrounding the veins (E), and in guard cells (F). (G) GFP fluorescence in epidermal cells after Agrobacterium-mediated transformation of Nicotiana benthamiana leaves with the CNGC20p:GFP construct. The bars represent 50 μm.

Figure 3

Salt regulation of CNGC19. (A-E) GUS staining of 18-day old plants carrying the CNGC19p:GUS construct, which were grown in the absence (A, D) or presence (B, E) of 150 mM NaCl for 7 days. (C, F) Luciferase activity in CNGC19p:LUC transformed plants. Promoter activity was determined as relative luciferase luminescence units (RLU) in shoots (C) and roots (F) normalized to the respective protein content. Roots of 12-day old plants were either untreated (black bars), or treated with 200 mM NaCl (blue bars) or 300 mM mannitol (white bars). Luciferase activities were determined after stress application at time points indicated. Data represent mean ± SEM (n = 3).

Figure 4

Salt regulation of CNGC20. CNGC20 promoter activity was monitored in 6-week old plants, using the luciferase reporter (A, B) or quantitative RT-PCR (C). (A) Luciferase activity in leaves from plants carrying the CNGC20p:LUC construct, at indicated time points after treatment of the roots with 200 mM NaCl (blue bars), 300 mM mannitol (white bars), tap water (yellow bars) or in untreated material (black bars). Data represent mean ± SEM (n = 3). (B) Petioles of detached leaves were put into Hoagland Medium (black bars), 200 mM NaCl-, 300 mM mannitol-, or tap water-containing solution and luciferase activity was quantified at time points indicated. Data represent mean ± SEM (n = 3). (C) CNGC20 transcript levels in detached leaves after treatment with 200 mM NaCl or tap water. Transcript levels are expressed relative to the respective actin control. Data represent mean ± SEM (n = 7-8). Color code in (B) and (C) as in (A).

Figure 5

Analysis of cngc19 and cngc20 T-DNA insertion lines. (A) Genomic organization of CNGC19 and CNGC20 and the respective T-DNA insertions for cngc19-1 (SALK line 027306) and cngc20-1 (SALK line 129133). Exons are shown in bold. (B) Absence of CNGC19 mRNA from cngc19-1 plants (lane 2), but presence in Col-0 (lane 1) and a backcrossed CNGC19 wild type (WT, lane 3). Upper traces: PCR result using CNGC19 gene-specific primers, which amplified a 348 bp fragment downstream of the T-DNA insertion, lower traces: Actin2 primers were used as a control. (C) Corresponding PCR analysis of the T-DNA insertion line cngc20-1 with cDNA from Col-0 (lane 1), cngc20-1 (lane 2) and a backcrossed CNGC20 wild type (WT, lane 3). CNGC20 gene-specific primers, which span a 310 bp fragment downstream of the T-DNA insertion, were used. No PCR fragment was amplified from cDNA from homozygous cngc20-1 (lane 2). (D) Unchanged root growth of cngc19-1 plants. Root length increase of cngc19-1 and wild type seedlings were measured during a 7-day growth period, starting 4 days after stratification. Mutant (orange bars) and wild type (black bars) plants grew vertically on half-strength MS agar plates in the absence (control) or presence of 10 μM ABA or 50 mM NaCl. Data represent mean ± SEM (n = 30). (E) Absence of a salt-dependent growth phenotype in cngc19-1 and cngc20-1. Photographs show representative plants from wild type, cngc19-1, and cngc20-1 after a 12-day growth period in the absence or presence of 75 mM NaCl. (F) Fresh weight of wild type (black circles), cngc19-1 (orange circles), and cngc20-1 (green circles) shoots after a 12-day growth period in the presence of 0, 25, 50, or 75 mM NaCl. Data represent mean ± SEM (n = 6). The dry weight did also not differ significantly between wild type and mutants (not shown). (G) K/Na content in shoots of plants shown in (E) and (F) as a function of the applied salt-concentration. Data represent mean ± SEM (n = 6). Color code as in (F).