The Relationship Between AtCaM1 and AtCaM4 and Salt Resistance

CaM, as the major Ca²⁺ sensor in plants, is involved in the responses of plants to a wide range of environmental stresses, including salt stress [41,42]. To determine which CaM isoform responds to salt, we first examined the expression of AtCaM1 (At5g37780), AtCaM2 (At2g27030), AtCaM3 (At3g56800), AtCaM4 (At1g66410), AtCaM5 (At2g41110), AtCaM6 (At5g21274), and AtCaM7 (At3g43810) using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Total RNA samples were prepared from wild-type seedlings treated with 50 mM NaCl. The AtCaM1 and AtCaM4 expression levels increased initially, reaching their highest values at 8 h (410 and 308% of the control level, respectively); they then decreased, but remained higher than in the control at 12 h (Fig 1A and 1D). The expression of the other genes showed no obvious regular variation (Fig 1B, 1C and 1E–1G). Thus, we reached the preliminary conclusion that out of all of the AtCaM genes investigated, AtCaM1 and AtCaM4, which encode the same protein [2], likely function in the response of Arabidopsis to salt.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Salt exposure influences AtCaM expression.

RT-qPCR analysis of AtCaM1 (A), AtCaM2 (B), AtCaM3 (C), AtCaM4 (D), AtCaM5 (E), AtCaM6 (F), and AtCaM7 (G) transcription in 7-day-old wild-type seedlings grown in 0.5× MS liquid medium with 50 mM NaCl for 0–12 h. The 18S rRNA was used as an internal control. The experiments were repeated three times with similar results. Each data point represents the mean ± standard deviation (SD, n = 3). Asterisks indicate a significant difference relative to 0 h (Student’s t-test, t-test, *P < 0.05 and **P < 0.01).

https://doi.org/10.1371/journal.pgen.1006255.g001

To confirm the role of AtCaM1 and AtCaM4 in salt stress tolerance, we compared the phenotypes of wild-type and mutant seedlings treated with or without salt stress. Due to the lack of an available T-DNA insertion mutant of AtCaM1 from the Arabidopsis Biological Resource Center (ABRC), we selected specific base sites (S1A Fig) to construct an artificial microRNA vector and then introduced it into wild-type and T-DNA insertion mutant cam4 (GABI_309E09) [26] plants to generate RNA interference (RNAi) transgenic lines. Next, four lines, cam1-1, cam1-2, cam1/4-1, and cam1/4-2, were selected for salt sensitivity analysis. No clear morphological difference was observed between 4-week-old wild-type and mutant plants under normal growth conditions (S1 Fig).

RT-qPCR analysis revealed dramatically reduced expression of AtCaM1 in cam1-1, cam1-2, cam1/4-1, and cam1/4-2 plants (7, 9, 13, and 23% of the control level, respectively), and nearly complete disruption of AtCaM4 in cam4, cam1/4-1, and cam1/4-2 plants (Fig 2A and 2B). However, deficiency in AtCaM4 slightly stimulated the expression of AtCaM1 (Fig 2A). Further, no obvious variation was found in the transcript levels of AtCaM2, AtCaM3, AtCaM5, AtCaM6, and AtCaM7 in these plants, indicating that a deficiency in AtCaM1 or AtCaM4 expression did not influence the expression of the other AtCaM genes (S2 Fig). Phenotypic observation indicated that the mutant seedlings were indistinguishable from wild-type seedlings under normal growth conditions. However, the effects of salt on the survival of the wild-type and mutant seedlings differed (Fig 2C). Following growth in medium containing 100 mM NaCl for 7 days [43], the survival ratios of the cam1-1, cam1-2, and cam4 mutants (55, 56, and 23%, respectively) were lower than that of wild type (79%). Double mutant (cam1/4-1 and cam1/4-2) seedlings showed greater sensitivity to salt stress than did the single mutant seedlings. Also, the survival ratio of the cam1/4-1 seedlings (12%) was lower than that of the cam1/4-2 seedlings (14%), consistent with their observed transcript levels (Fig 2D).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. AtCaM1 and AtCaM4 contribute to salt tolerance.

(A, B) RT-qPCR analysis of AtCaM1 (A) and AtCaM4 (B) transcription in wild-type and mutant plants. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to Columbia (Col) (Student’s t-test, t-test, *P < 0.05, **P < 0.01, and ***P < 0.001). (C) Salt stress sensitivity of 7-day-old seedlings grown in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. (D) Survival ratios of the seedlings after salt treatment. Those seedlings with still green cotyledons were scored as survivors. Each data point represents the mean ± standard error (SE, n = 30). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01). (E) RT-qPCR analysis of salt-responsive gene expression in wild-type, cam1/4-1, and cam1/4-2 seedlings. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). The numbers at the top indicate the fold change in mRNA level after the salt challenge.

https://doi.org/10.1371/journal.pgen.1006255.g002

Next, we identified unique bases in AtCaM1 and AtCaM4 through a comparison to other CaM genes (S3A Fig) in order to produce RNAi transgenic lines. Two lines, cam1/4-3 and cam1/4-4, were selected for analysis. No obvious morphological difference was observed among 4-week-old wild-type and mutant plants under normal growth conditions (S3B Fig). RT-qPCR analysis showed that the transcript levels of AtCaM1 and AtCaM4 were greatly decreased in the cam1/4-3 and cam1/4-4 plants, especially in cam1/4-3 (S3C and S3D Fig). Under salt stress, the survival ratios of the cam1/4-3 and cam1/4-4 seedlings were significantly reduced (31 and 25%, respectively) compared with that of wild-type seedlings; the greater reduction in the cam1/4-3 plants mirrors the observed decrease in mRNA expression (S4 Fig). We next detected salt-induced genes in Arabidopsis and found that their expression was dramatically induced by NaCl in wild-type, but to a lesser extent in cam1/4-1 and cam1/4-2, seedlings (Fig 2E), further indicating that AtCaM1 and AtCaM4 function in salt tolerance.

Seven distinct genes in Arabidopsis encode AtCaMs [2]; we utilized T-DNA insertion mutants of cam2 (SALK_114166) and cam3 (SALK_001357), with disrupted expression of AtCaM2 and AtCaM3, respectively [26], to examine the roles of other CaM genes in salt tolerance. Under normal growth conditions, no obvious phenotypic difference was detected among the wild-type, cam2, and cam3 seedlings. The same result was obtained following salt treatment: the survival ratios of cam2 and cam3 mutant seedlings were similar to that of wild-type seedlings (S5 Fig). These observations suggest that other AtCaM genes do not function in salt resistance.

Effect of AtCaM4 on the Salt Sensitivity of cam1/4-1 Mutant Plants

To further test whether the salt sensitivity of the mutant plants resulted from the loss of AtCaM1 and AtCaM4, AtCaM4 complementation lines (cam1/4-1 + AtCaM4, 4COM), and AtCaM1 and AtCaM4 overexpression lines (ecotype Columbia [WT] + AtCaM1, 1OE; ecotype Columbia [WT] + AtCaM4, 4OE) were generated and confirmed by RT-qPCR and reverse transcription polymerase chain reaction (RT-PCR), respectively (S6, S8 and S9 Figs). In the AtCaM4 complementation lines (4COM1 and 4COM2), the AtCaM1 mRNA level was rescued to a near wild-type level, suggesting ineffective RNAi (S6A and S6B Fig). Under normal growth conditions, none of the transgenic lines showed a mutant phenotype compared with wild type (S6, S8 and S9 Figs). When subjected to salt stress for 7 days, the AtCaM4 complementation lines exhibited enhanced survival, similar to wild type (S7 Fig), providing genetic proof of the involvement of AtCaM1 and AtCaM4 in salt resistance.

However, no significant morphological difference was detected between the wild-type and AtCaM1- and AtCaM4-overexpressing lines (1OE1 and 1OE2, and 4OE1 and 4OE2, respectively) under conditions of salt stress (S8C and S9C Figs).

Effects of Salt Stress on NO Accumulation and Survival in Wild-Type and Mutant Seedlings

In mammalian cells, CaM is thought to bind and thus activate NOS isozymes to stimulate NO production. NO, as a signaling molecule, plays an important role in the salt stress signaling pathway in Arabidopsis seedlings [32]. Thus, it is reasonable to assume that AtCaM1 and AtCaM4 mediate salt resistance by regulating NO metabolism.

Accordingly, we examined intracellular NO formation in wild-type, cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 plants and in two AtCaM4 complementation lines at the seedling stage. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) was selected for use as a fluorescent probe for NO because it is highly specific for NO and does not react with other reactive oxygen species. DAF-FM DA permeated the membrane and was transformed by intracellular esterases into 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), which reacts with NO to create a highly fluorescent triazole compound [44]. A special NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) decreased the fluorescence density depending on its concentration, indicating DAF-FM DA was the special probe for NO (S10 Fig). Fluorescence analysis revealed that the NO levels were relatively stable in the seedlings under normal growth conditions. However, the NO level was remarkably increased in the presence of NaCl and varied depending on the expression of AtCaM1 and AtCaM4; it was increased by 260% in wild-type seedlings, which is greater than in the cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 mutant seedlings (the lowest values were obtained from the cam1/4-1 and cam1/4-2 double mutants; 72 and 81%, respectively). However, the NO level was nearly completely rescued in the AtCaM4 complementation lines (Fig 3A and 3B). By combining these data with the results of our salt tolerance analysis (Fig 2), we might conclude that the salt sensitivity of cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 was due to the low endogenous NO level.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. AtCaM1 and AtCaM4 are positive regulators of salt-mediated NO accumulation.

(A) NO accumulation in the roots of 7-day-old wild-type, cam1-1, cam1-2, cam4, cam1/4-1, cam1/4-2, 4COM1 and 4COM2 seedlings grown in 0.5× MS liquid medium with or without 100 mM NaCl for 24 h was detected by DAF-FM DA staining. The experiments were repeated three times with similar results. Bar = 50 μm. (B) Relative DCF fluorescence densities in the roots. Each data point represents the mean ± SE (n = 20). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01). (C) Salt stress sensitivity of 5-day-old seedlings grown in 0.5× MS medium with or without 100 mM NaCl supplemented with 50 μM GSNO, 100 μM c-PTIO or 150 μM L-NAME for another 2 days. The experiments were repeated three times with similar results. (D) Survival ratios of the seedlings after salt treatment. Each data point represents the mean ± SE (n = 30). Asterisks indicate a significant difference relative to NaCl (Student’s t-test, *P < 0.05). (E) Root lengths of the seedlings with or without 100 mM NaCl. Each data point represents the mean ± SE (n = 30). *P < 0.05 (Student’s t-test).

https://doi.org/10.1371/journal.pgen.1006255.g003

To further confirm the effects of NO on the salt sensitivity of the mutant plants, we examined the effects of NO donor and inhibitors on their survival. Exogenous application of 50 μM GSNO, as NO donor, increased the root lengths of the plants under both normal and high-salt conditions, and it increased the survival ratios of the mutant seedlings under high-salt conditions. Whereas exogenous application of 100 μM cPTIO or 150 μM N^G-nitro-L-arginine-methyl ester (L-NAME, a NO synthase inhibitor) showed the adverse effects on them (Fig 3C to 3E), indicating that NO acts as a downstream mediator of AtCaM1 and AtCaM4 in salt tolerance.

Examination of Direct Binding Between AtCaM4 and GSNOR

CaM, as a central signaling molecule, likely confers salt tolerance by binding directly to a specific target protein. Therefore, we next sought to identify interacting proteins of AtCaM4 (and AtCaM1, omitted) to gain insight into the roles of CaM in salt signaling.

A number of experimental approaches have been employed in previous studies to identify CaM-interacting proteins [13, 45]. Herein, we carried out experiments to explore what AtCaM4-binding protein(s) regulates NO homeostasis in salt-treated plants. According to a structural analysis conducted using WebLab ViewerLite (Accelrys, San Diego, CA), GSNOR (among NO metabolism-related proteins; see the Introduction) harbors important binding elements for the paired EF hands in AtCaM4 (S11 Fig). Thus, we performed three assays to test whether AtCaM4 is a substrate of GSNOR. A bimolecular fluorescence complementation (BiFC) assay [46] in tobacco (Nicotiana tabacum) leaves showed that negative combinations, including CaM4-YN/YC (in which the N-terminal followed by the C-terminal half of yellow fluorescent protein [YFP] was fused to CAM4) and CaM4-YC/YN (in which the C-terminal followed by the N-terminal half of YFP was fused to CAM4), did not produce any detectable fluorescence, while the co-expression of CaM4-YN (in which the N-terminal half of YFP was fused to CAM4) and GSNOR-YC (in which the C-terminal half of YFP was fused to GSNOR) or CaM4-YC (in which the C-terminal half of YFP was fused to CAM4) and GSNOR-YN (in which the N-terminal half of YFP was fused to GSNOR) produced strong YFP signals, which were mainly localized to the plasma membrane of the cotransformed tobacco epidermal cells (Fig 4A). Furthermore, we performed a domain deletion analysis (see SMART, http://smart.embl-heidelberg.de) by overlay assay (Fig 4B). To map the CaM-binding domain of GSNOR, we created a series of GSH S-transferase (GST) fusion constructs containing the full-length cDNA and two serial deletion mutants. Our results show that GST-tagged full-length GSNOR, the N-terminal fragment of GSNOR, and the C-terminal fragment of GSNOR all interacted with CaM-His when Ca²⁺ was present in the medium, whereas GST alone did not interact with AtCaM4. However, the binding became weaker in the presence of 0.1 mM EGTA (a Ca²⁺ chelator) (Fig 4C), indicating that AtCaM4 binds to GSNOR at multiple important regions (S11 Fig) in a Ca²⁺-dependent manner (Fig 4B). The interaction of CaM4 and GSNOR in vivo was confirmed using co-immunoprecipitation (co-IP) assays. Proteins were extracted from Arabidopsis harboring GSNOR and CaM4_pro:CaM4-green fluorescent protein (GFP) constructs and used for co-IP assays. As shown in Fig 4D, the immunoprecipitation of CaM4 with anti-GFP agarose conjugate yielded a co-IP band corresponding to GSNOR that was labeled with anti-GSNOR antibodies; moreover, the signal intensity was increased by exposure to NaCl. Taken together, these in vivo and in vitro protein-protein interaction assays indicate a direct interaction between CaM4 and GSNOR.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. AtCaM4 binds to GSNOR in response to salt stress.

(A) In planta BiFC assay of the CaM4 interaction with GSNOR in tobacco leaves. The C-terminal half of YFP was fused to CAM4 (CaM4-YC) and GSNOR (GSNOR-YC), and the N-terminal half of YFP was fused to GSNOR (GSNOR-YN) and CAM4 (CaM4-YN). The expression of CaM4-YC/YN and CaM4-YN/YC was used as a control. Bar = 50 μm. (B) Model of the GSNOR N- and C-terminal deletions for the overlay assay. (C) In vitro overlay assay of the CaM4 interaction with GSNOR. GST and GST-GSNOR fusion proteins were expressed in E. coli. Recombinant proteins were analyzed by Western blotting (WB) using anti-GST antibodies. The overlay assay was performed using CaM4-His in the presence of 0.1 mM CaCl₂ or 0.1 mM EGTA. (D) Co-IP assays showing the interaction between CaM4 and GSNOR. CaM4-GFP was expressed from the native CaM4 promoter. Extracted proteins were incubated with anti-GFP agarose beads. Total and immunoprecipitated proteins were analyzed by immunoblotting using anti-GSNOR and -GFP antibodies.

https://doi.org/10.1371/journal.pgen.1006255.g004

What is the effect of this binding on GSNOR activity? We measured the GSNOR activity in wild-type, cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 plants as well as in two AtCaM4 complementation lines with total protein and purified GSNOR protein from the seedlings. Our data indicate no clear difference among the seedlings in terms of GSNOR activity under normal conditions. However, the level of GSNOR activity from total protein was greatly increased by NaCl in the cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 mutant seedlings (it was highest for cam1/4-1 and cam1/4-2), whereas it was only slightly increased in the wild-type, 4COM1, and 4COM2 seedlings (Fig 5A). Immunoblotting showed that GSNOR expression did not vary noticeably in the seedlings treated with and without salt (Fig 5B), indicating that AtCaM4 inhibited GSNOR activity directly but did not greatly influence its expression. Levels of NO-related metabolite S-nitrosothiols (SNOs) in vivo are controlled by NO synthesis and by GSNO turnover, which is mainly performed by GSNOR [47]. Thus, we measured the variation of SNO content in the seedlings after NaCl treatment. Our data indicate that total SNO levels were only slightly increased in the cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 mutant seedlings (it was lowest for cam1/4-1 and cam1/4-2), whereas it was greatly increased in the wild-type, 4COM1, and 4COM2 seedlings (Fig 5C), in an opposite changing manner as GSNOR activity (Fig 5A), implying that increased GSNOR activity in the mutant seedlings (especially for cam1/4-1 and cam1/4-2) inhibited SNO accumulation. Exogenous application of CaM4-GST fusion inhibited GSNOR activity in a concentration-dependent manner, whereas exogenous application of EGTA enhanced it in the cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 mutant seedlings (Fig 5D), providing the straight evidence for AtCaM4 inhibition of GSNOR activity. The activity of the same content of purified GSNOR protein from these seedlings (Fig 5F) also showed the same changing manner (Fig 5E) as that of total protein (Fig 5A), further indicating that AtCaM4 inhibition of GSNOR activity was not due to the variation of GSNOR expression. Together, these data show that AtCaM4 bound to GSNOR directly and influenced its activity under salt stress; thus, GSNOR is a target of AtCaM4 in the salt signaling pathway.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. AtCaM4 decreases GSNOR activity in response to salt stress.

(A, E) GSNOR activity of total protein (A) or purified protein (E) from 7-day-old wild-type, cam1-1, cam1-2, cam4, cam1/4-1, cam1/4-2, 4COM1 and 4COM2 seedlings grown in 0.5 × MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to 0 mM NaCl (Student’s t-test, *P < 0.05 and **P < 0.01). (B) Immunoblot analysis of GSNOR expression in the seedlings. Tubulin was used as an internal control. The experiments were repeated three times; the results indicate similar trends in protein accumulation. (C) SNO content in the seedlings grown in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). The numbers at the top indicate the fold change in SNO level after the salt challenge. (D) GSNOR activity in 7-day-old cam1-1, cam1-2, cam4, cam1/4-1, and cam1/4-2 seedling after addition of 0.1 mM EGTA, 0.02 or 0.04 mM CaM4-GST in the reaction mixture. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to 100 mM NaCl (Student’s t-test, *P < 0.05). (F) Immunoblot analysis of purified GSNOR from total protein with anti-GSNOR. gsnor seedlings was used as a negative control. The experiments were repeated three times; the results indicate similar trends in protein accumulation.

https://doi.org/10.1371/journal.pgen.1006255.g005

Unexpectedly, an in vitro experiment using CaM4-His and GST-GSNOR fusions, which were expressed in Escherichia coli (E. coli) and purified (S12A Fig), showed that CaM4 had no obvious effect on GSNOR activity (S12B Fig). A plausible explanation for this strange phenomenon could be the need of a proper conformation or additional posttranslational modification of both proteins.

GSNOR Negatively Regulates the NO Level and Salt Tolerance in Plants

An analysis by RT-qPCR indicated that GSNOR expression was slightly stimulated by salt treatment (S13A Fig). β-Glucuronidase (GUS) staining of GSNOR_pro:GUS transgenic plants showed that the GSNOR promoter drove expression ubiquitously (S13B Fig), suggesting that it has extensive activities in plants. To determine the subcellular localization of GSNOR and AtCaM4, 35S_pro:GSNOR-GFP and 35Spro:CaM4-GFP fusions were introduced into tobacco leaves, respectively; similarly, both of GFP fluorescence was found to be localized mainly in the cell membrane, cytoplasm, and nucleus (S13C and S14 Figs).

GSNOR is believed to be an important and widely utilized regulatory component of NO homeostasis in plant resistance protein signaling networks [45, 48–52]. The T-DNA mutant gsnor (CS66012, also named hot5-2 [53]), which carries an insertion in exon 1, was obtained from the ABRC. To analyze the physiological role of GSNOR in plants under salt stress, we assayed the NO level and salt tolerance in wild-type and gsnor plants, and in two complementation lines (gsnor + GSNOR, 2COM) and two overexpression lines (ecotype Columbia [WT] + GSNOR, 2OE), which were verified by RT-qPCR and immunoblot analyses for transcript and protein accumulation (Fig 6A). Thereafter, fluorescence analysis revealed no obvious change in NO among the seedlings under normal conditions. However, the NO level increased clearly under 100 mM NaCl treatment and was far higher in the gsnor seedlings than in the wild-type seedlings, fully restored in the two gsnor complementation lines (2COM1 and 2COM2), and reversed in the two gsnor-overexpression lines (2OE1 and 2OE2) (Fig 6B and 6C). Additionally, the gsnor seedlings were small under both normal and high-salt conditions; however, their survival ratio was 14% higher than that of wild-type seedlings when grown on NaCl-containing medium. This situation was restored in the two complementation lines and reversed in the two overexpression lines (Fig 6D and 6E), implying that GSNOR contributes to salt sensitivity via inhibition of the endogenous NO level in plants. Simultaneously, the root length of the gsnor seedlings was less reduced compared to that of wild-type seedlings in the existence of NaCl. This situation was partially restored in the complementation and overexpression lines depending on their internal NO levels (Fig 6F), implying NO stimulation of root growth.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. GSNOR acts as a negative regulator of salt tolerance.

(A) RT-qPCR and immunoblot analyses of GSNOR expression in wild-type, gsnor, 2COM1, 2COM2, 2OE1 and 2OE2 plants. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05, **P < 0.01, and ***P < 0.001). (B) NO accumulation in the roots of 7-day-old seedlings. The experiments were repeated three times with similar results. Bar = 50 μm. (C) Relative DCF fluorescence densities in the roots. Each data point represents the mean ± SE (n = 20). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01). (D) Salt stress sensitivity of the seedlings in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. (E) Survival ratios of the seedlings after salt treatment. Each data point represents the mean ± SE (n = 30). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01). (F) Root lengths of the seedlings with or without 100 mM NaCl. Each data point represents the mean ± SE (n = 30). The numbers at the top indicate the fold change in root length after the salt challenge.

https://doi.org/10.1371/journal.pgen.1006255.g006

The Relationship between AtCaM4 and GSNOR in Response to Salt Stress

To examine the underlying mechanism of AtCaM1/4- and GSNOR-induced salt tolerance in Arabidopsis, we obtained GSNOR-overexpressing transgenic lines in a cam4 background (2OE/cam4) and compared their NO levels and survival. RT-qPCR revealed stronger exogenous GSNOR expression in the 2OE1/cam4 and 2OE2/cam4 lines than in wild type (Fig 7A). These two lines did not show obvious variation in the level of NO compared with cam4 under normal growth conditions. However, under 100 mM NaCl treatment, the NO levels and survival ratios of the two transgenic lines were lower than those of cam4 (survival ratios of 17 and 15% versus 23%, respectively), indicating that overexpression of GSNOR further reduced the NO level and strengthened the salt sensitivity of the cam4 mutant (Fig 7B–7E).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. GSNOR overexpression inhibits NO accumulation and enhances salt sensitivity in cam4.

(A) RT-qPCR analysis of AtCaM4 and GSNOR transcription in wild-type, cam4, 2OE1/gsnor and 2OE2/gsnor plants. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05, **P < 0.01, and ***P < 0.001). (B) NO accumulation in the roots of 7-day-old seedlings. The experiments were repeated three times with similar results. Bar = 50 μm. (C) Relative DCF fluorescence densities in the roots. Each data point represents the mean ± SE (n = 20). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01). (D) Salt stress sensitivity of the seedlings in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. (E) Survival ratios of the seedlings after salt treatment. Each data point represents the mean ± SE (n = 30). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01).

https://doi.org/10.1371/journal.pgen.1006255.g007

We also compared the NO levels and survival of wild-type, cam4, gsnor, and cam4gsnor double mutant (deficient in CaM4 and GSNOR transcription) seedlings (Fig 8A). Under normal growth conditions, the NO levels in the seedlings were not obviously different; however, they were increased by NaCl. Surprisingly, the NO level in cam4gsnor was increased compared with that in cam4 and similar to that in gsnor (Fig 8B and 8C). Regardless of whether NaCl was present, the cam4gsnor double mutant seedlings showed reduced shoot and root growth, like the gsnor seedlings. After salt treatment, the survival ratio changed in a manner similar to the NO level: the survival ratio of cam4gsnor was higher than that of wild type and similar to that of gsnor (Fig 8D and 8E).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. GSNOR disruption enhances NO accumulation and inhibits salt sensitivity in cam4.

(A) RT-qPCR analysis of AtCaM4 and GSNOR transcription in wild-type, cam4, gsnor, and cam4gsnor plants. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05, and **P < 0.01). (B) NO accumulation in the roots of 7-day-old seedlings. The experiments were repeated three times with similar results. Bar = 50 μm. (C) Relative DCF fluorescence densities in the roots. Each data point represents the mean ± SE (n = 20). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05). (D) Salt stress sensitivity of the seedlings in 0.5× MS medium with or without 100 mM NaCl. The experiments were repeated three times with similar results. (E) Survival ratios of the seedlings after salt treatment. Each data point represents the mean ± SE (n = 30). Asterisks indicate a significant difference relative to Col (Student’s t-test, *P < 0.05 and **P < 0.01).

https://doi.org/10.1371/journal.pgen.1006255.g008

Taking these results together, we might conclude that AtCaM1 and AtCaM4 confer salt tolerance by mediating NO accumulation through GSNOR.

AtCaM4 and GSNOR Modulate Ion Absorption in Arabidopsis Seedlings

When plants are exposed to NaCl, cellular ion homeostasis may be impaired. Under saline conditions, tolerant plants typically maintain high K⁺ and low Na⁺ levels in the cytosol via the compartmentalization of Na⁺ into vacuoles and/or extrusion to the external medium and the accumulation of K⁺ in the cytoplasm [54,55].

It was previously reported that NO functions as a second messenger in reestablishing ion homeostasis to resist salt stress in reed calluses (P. communis Trin.) [31] and Arabidopsis seedlings [32]. In the present study, we tested the effects of CaM4-GSNOR on the NO-mediated regulation of ion absorption in Arabidopsis seedlings exposed to excessive salt. The inhibition of K⁺ absorption and stimulation of Na⁺ absorption, as well as a decreased K⁺/Na⁺ ratio, were observed in cam1-1, cam4, and cam1/4-1 mutant plants compared with wild type (especially in cam1/4-1), though the opposite situation was detected in the gsnor mutant. This trend was intensified in the GSNOR-overexpressing transgenic line 2OE1/cam4 but was partially rescued in the cam4gsnor double mutant compared to cam4 (Fig 9), suggesting that AtCaM4 enhances K⁺ absorption and inhibits Na⁺ absorption through GSNOR in plants under salt stress.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 9. AtCaM4 influences ion accumulation in response to salt stress through GSNOR.

The (A) Na⁺ content, (B) K⁺ content, and (C) Na⁺/K⁺ ratio in 7-day-old wild-type, cam1-1, cam4, cam1/4-1, 2OE1/cam4, and cam4gsnor seedlings grown in 0.5× MS liquid medium with or without 100 mM NaCl for 24 h. The experiments were repeated three times with similar results. Each data point represents the mean ± SD (n = 3). Asterisks indicate a significant difference relative to Col in each group (Student’s t-test, *P < 0.05 and **P < 0.01).

https://doi.org/10.1371/journal.pgen.1006255.g009

AtCaM1 and AtCaM4 Confer Salt Resistance in Arabidopsis Seedlings

CaMs are predicted to function in response to a rise in the cytoplasmic concentration of Ca²⁺ in many physiological processes in plants and animals [20]. Considering the many processes in which they are involved, their diverse subcellular localization patterns, and their assorted responses to various environmental stimuli, each CaM isoform may have a specific function [56]; for example, AtCaM3 is involved in the induction of cold- and heat-responsive genes [25, 26], while AtCaM7 is involved in the induction of light-responsive genes [24]. Currently, the role of CaM in the salt signaling pathway is elusive, greatly limiting our knowledge of plant adaptation to salt stress.

The present study demonstrates the involvement of AtCaM1 and AtCaM4 in salt stress signaling. In salt-treated plants, AtCaM1 and AtCaM4 act as second messengers; they bind GSNOR and reduce its activity so as to elevate the endogenous NO level and reestablish cellular ion homeostasis. Thus, AtCaM1 and AtCaM4 promote salt resistance in Arabidopsis seedlings.

CaM is regarded as a signaling molecule that contributes to salt tolerance; however, its signaling pathway is unknown. To address this, two key elements must be identified: salt-responsive CaM isoforms and their corresponding target protein(s). In Arabidopsis, only seven genes encode real CaM proteins [2]. Therefore, we first examined their expression under salt stress. RT-qPCR analysis showed that AtCaM1 and AtCaM4 transcript accumulation varied regularly under saline conditions (Fig 1). The disruption of AtCaM1 and AtCaM4 resulted in poor salt tolerance in cam1-1, cam1-2, cam4, and double mutant plants depending on their transcript levels (Fig 2). The knockdown of both AtCaM1 and AtCaM4 exacerbated the salt sensitivity of the plants compared to the knockdown of only AtCaM1 (S4 Fig), suggesting the positive role of these genes in salt resistance. Rescued AtCaM4 expression in AtCaM1/4-1 nearly totally restored the salt sensitivity of the plants (S7 Fig), providing genetic support for this hypothesis.

A loss of AtCaM1 and AtCaM4 impaired salt-responsive signaling, as evidenced by the significant decrease in the fold changes of salt-induced genes in RNAi plants compared with wild-type plants (Fig 2E). Taken together, these observations suggest that AtCaM1 and AtCaM4 each contribute to salt resistance and that their functions do not overlap.

Genetic studies have been invaluable in improving our understanding of the role of CaMs in angiosperms. However, the high level of sequence identity among members of the CaM family and the likelihood of functional redundancy have complicated this approach. Thus, we examined the expression and function of other CaM isoforms. Our results indicate that a loss of AtCaM1 and AtCaM4 transcription did not influence the expression of other CaM isoforms in plants (S2 Fig). Further, the deletion of AtCaM2 and AtCaM3 did not obviously modulate plant salt sensitivity (S5 Fig). These data suggest that AtCaM1 and AtCaM4 are the salt-responsive CaM isoforms; other CaM isoforms may not affect salt tolerance in the seedlings.

We also observed a strange phenomenon: the overexpression of AtCaM1 or AtCaM4 did not enhance salt tolerance (S8 and S9 Figs), possibly due to the natural high abundance of AtCaMs and the amount of interacting protein (i.e. GSNOR; Fig 5B).

AtCaM4 Regulates NO Production in Response to Salinity

In mammalian cells, CaM, a ubiquitous 17-kDa cytosolic protein, is a major cellular Ca²⁺ sensor that rapidly regulates intracellular processes through its coordinated activity with more than 50 intracellular proteins, including NOS [57]. Previous studies have shown that CaM participates in a wide variety of processes, including neurotransmission, vasodilation, and immune defense, by regulating the production of NO through NOS [58]. In plants, a NO synthesis-related enzyme is stimulated by salt to enhance the internal NO level and initiate plant defensive reactions [31,32]. Thus, we hypothesized that CaMs mediate the NO level to initiate plant responses to salt stress.

As shown in Fig 3A and 3B, the internal NO level increased depending on the expression levels of AtCaM1 and AtCaM4 in response to salt stress. Also, the NO donor GSNO increased the salt tolerance of AtCaM1 and AtCaM4 mutant plants to a level near that of wild type but the NO inhibitors cPTIO and L-NAME decreased it (Fig 3C and 3D), providing further proof for this hypothesis. NO, which functions as an important messenger in multiple biological processes in plants, is induced by numerous biotic and abiotic stresses to mediate resistance responses, however, it relationship with CaM remained to be elucidated. Therefore, our study implies a common defense system in plant. Among the three major families of Ca²⁺ sensors in terrestrial plants, only CaMs co-exist in plants and animals. Thus, these findings support the idea of a common pathway of this defense system in higher eukaryotes.

GSNOR Is the Target Protein of AtCaM4 in Response to Salinity

Identifying the specific target (or substrate) of a CaM isoform is a key step in understanding the functions of CaM in plant signaling. The specific target of a CaM protein must exhibit two functional characteristics: it must bind to CaM, and its activity must subsequently change. A transient expression study with protoplasts indicated that the Ca²⁺/CaM complex functions as a negative regulator of the activity of the rice (Oryza sativa) CAMTA/SR protein Os-CBT [59]. The expression of two CaM-binding TFs in bean is induced in response to incompatible pathogens and elicitors of plant defense responses, suggesting a role for CaM-binding TFs in plant defense [60]. A loss-of-function mutant of a CaM-binding phosphatase (PP7) exhibited reduced heat tolerance, whereas its overexpression, which increased heat shock protein expression, conferred thermotolerance [61]. Since this phosphatase interacts with a heat shock TF (HSF1), it is likely that Ca²⁺/CaM modulates the activity of HSF1 through PP7.

Accordingly, we first sought to identify AtCaM4-binding proteins involved in NO metabolism in plants under salt stress. In mammalian cells, CaMs bind NOS isozymes directly. A bioinformatics analysis did not predict the interaction of AtCaM4 and NO synthesis-related proteins, including NO-associated protein 1, nitrate reductase 1, and nitrate reductase 2 [34]; however, it suggested the binding of AtCaM4 to GSNOR (S11 Fig), which degrades GSNO, a stable and mobile NO pool, to reduce the overall accumulation of NO in plant cells. In vivo and in vitro assays revealed direct binding between AtCaM4 and GSNOR (Fig 4A–4D), which could be inhibited by a Ca²⁺ chelator, indicating that this binding was initiated by enhancement of the cytoplasmic Ca²⁺ concentration. N- and C-terminal fragments of GSNOR interacted with CaM4, indicating that two or more elements in GSNOR bind the paired EF hands in AtCaM4 (Fig 4C), consistent with the predicted model (S11 Fig). Further, the binding of AtCaM4 to GSNOR was reinforced in the presence of NaCl (Fig 4D), indicating a possible role in the response of plants to salt stress.

Next, we showed that CaM4 inhibited GSNOR activity according to its expression level but had no great effect on GSNOR expression (Fig 5). We also found that deficiency in CaM4 led to slightly lower GSNOR mRNA level (Fig 8A), implying no great effect of CaM4 on GSNOR expression under normal conditions. These data suggest that AtCaM4 directly binds to GSNOR and subsequently inhibits its activity, indicating that GSNOR is a specific target of AtCaM4 in the salt signaling pathway.

CaM4 through GSNOR Regulates NO Production in Salt Resistance

NO bioactivity is controlled by NO synthesis and degradation, which is mainly performed by GSNOR [62,63]. Additionally, the NO system is mainly regulated by the breakdown of GSNO by GSNOR, which is conserved from bacteria to humans [62]. In Arabidopsis, GSNOR, previously known as GSH-dependent formaldehyde dehydrogenase or class III alcohol dehydrogenase (ADH) due to its interaction with primary alcohols, is encoded by a single copy gene, GSNOR [35]. GSNOR is an important and widely utilized component of resistance protein signaling networks that controls NO accumulation. However, its role in the salt signaling pathway is not yet clarified.

As shown in Fig 6, gsnor mutant plants displayed a higher NO level and enhanced salt tolerance compared to wild-type plants; this phenotype was restored in two complementation lines, and was reversed in two overexpression lines, implying that GSNOR is a negative regulator of salt tolerance according to its inhibition of NO accumulation (Fig 6). We hypothesize that AtCaM4, by binding to and reducing the activity of GSNOR, can enhance the NO level in salt-stressed plants. In the existence of NaCl, NO exhibited positive effects on root growth (Figs 3E and 6F), which should be due to its action on stem cell niche homeostasis through interaction with auxin [64]. However, high levels of NO reduce auxin transport and response by a PIN1-dependent mechanism, and root meristem activity is reduced concomitantly [65], implying that NO through auxin regulates root growth in a concentration-dependent manner.

To further verify the relationship between CaM4-GSNOR and NO in salt signaling, we obtained GSNOR-overexpression transgenic lines in a cam4 background and cam4gsnor double mutant plants. Surprisingly, GSNOR overexpression reduced both the internal NO level and survival of cam4 plants, indicating that GSNOR acts downstream of AtCaM4 and inhibits NO accumulation (Fig 7). The deletion of GSNOR enhanced the salt tolerance of cam4 plants accompanied by enhancement of the NO level (Fig 8). One plausible explanation for this is that GSNOR deletion increased the supply of NO in the absence of AtCaM4 so as to affect other different molecular components (i.e. S-nitrosylation of specific proteins) to increase salt resistance, suggesting that CaM4-GSNOR stimulates internal NO accumulation under saline conditions.

The Mechanism Underlying the Effect of AtCaM4 Through GSNOR in Salt-Stressed Plants

When plants are exposed to high concentrations of Na⁺, the excess Na⁺ ions tend to substitute for K⁺ due to physicochemical similarities between Na⁺ and K⁺, leading to plant dysfunction [66]. The ability to control net Na⁺ influx into the cytoplasm and to maintain a minimal Na⁺/K⁺ ratio in the cytoplasm is of great importance in determining plant responses to salinity [67]. NO alleviates salt toxicity in reed [31] and maize [68] through the up-regulation of H⁺-ATPase activity in the plasma membrane and vacuolar membrane, resulting in Na⁺ efflux into the apoplast and vacuole. As a glycophytic species, Arabidopsis is sensitive to moderate levels of NaCl and accumulates a significant amount of Na⁺ when exposed to salinity [19]. NO is associated with salt tolerance in Arabidopsis via attenuation of the NaCl-induced increase in the Na⁺/K⁺ ratio [32].

In the present study, the Na⁺/K⁺ ratio increased with the loss of AtCaM1 and AtCaM4 expression under saline conditions, whereas it decreased in the gsnor mutant. This situation was enhanced in the GSNOR-overexpression lines but partially rescued in the cam4gsnor double mutant compared to cam4 plants, indicating that AtCaM1 and AtCaM4 influence ion absorption through GSNOR (Fig 9).

The present data indicate that AtCaM1 and AtCaM4 regulate ion absorption and affect salt resistance in plants by increasing the cellular level of NO through binding to and inhibiting the activity of GSNOR (Fig 10). Their findings suggest that this plant defensive pathway could share a common evolutionary origin with animals. NO was even reported to control its own generation and scavenging by modulating nitrate assimilation and GSNOR1 activity [69], indicating a feedback inhibition between GSNOR and NO in plants.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 10. The Ca²⁺/CaM/GSNOR cascade mediates salt resistance in arabidopsis seedlings.

(A) Under normal conditions, calcium channel closure limits Ca²⁺ entry so as to prevent CaM binding and Ca²⁺/CaM signaling transduction. The NO level was kept relatively stable by GSNOR. (B) During salt exposure, calcium channels are activated and open. Consequently, the formed Ca²⁺/CaM complex interacts directly with GSNOR and inhibits its activity, thereby stimulating NO accumulation and ion homeostasis to confer salt resistance. Thick arrows indicate normal pathways; dotted arrows show weaker processes; a straight line shows repressive effect. The question mark indicates an incomplete complex.

https://doi.org/10.1371/journal.pgen.1006255.g010

Plant Materials and Growth Conditions

Arabidopsis (A. thaliana ecotype Columbia) and tobacco (N. tabacum) were used in this study. The T-DNA insertion line gsnor (CS66012) was obtained from the ABRC (http://www.arabidopsis.org/abrc/). The other lines used in this study, cam2 (SALK_114166), cam3 (SALK_001357), and cam4 (GABI_309E09), were obtained from Drs. Daye Sun and Sujuan Cui (Hebei Normal University). The cam4gsnor double mutant was obtained by crossing. RNAi constructs for AtCaM1 and AtCaM4 were made and corresponding RNAi plants were produced (the primers used are shown in S1 Table).

Seeds were surface-sterilized in 2% (v/v) sodium hypochlorite for 1 min and then washed thoroughly with water. The sterilized seeds were plated on 0.5× Murashige and Skoog (MS) medium containing 1.5% sucrose and 0.3% agar and kept at 4°C in the dark for 2 days. The plants were then transferred to a growth chamber set at 22°C and 120 μmol/m²s on a 16-h day/night cycle.

Tobacco seeds were planted in potting mix (2:1 [v/v] rich soil:vermiculite) and kept in a growth chamber at 23°C with illumination at 120 μmol/m²s with a 16-h daily light period. After 3 weeks of growth, the plants were used for transformation.

Stress Treatments

After germination, seedlings from each line were carefully transferred to a fresh MS agar plate supplemented with 100 mM NaCl. After 7 days of growth on the treatment medium, those seedlings with still green cotyledons were scored as survivors.

For all chemical treatments, 1 ml of 50 μM GSNO, 100 μM cPTIO or 150 μM L-NAME (Sigma-Aldrich, St. Louis, MO) was sprayed onto the leaf surfaces of 5-day-old seedlings after filter-sterilization for 48 h. Control seedlings were treated with water.

RT-PCR and RT-qPCR

Total RNA (500 ng) was isolated using a PrimeScript RT reagent kit (Takara Bio Inc., Otsu, Japan) for first-stand complementary DNA synthesis. RT-PCR analyses of AtCaM1 and AtCaM4 transcription were performed using a Takara RNA PCR (Avian Myeloblastosis Virus) kit version 3.0 (Takara Bio Inc.) with gene-specific primers (S1 Table). RT-qPCR analyses of gene expression were done using an ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA) with SYBR Premix Ex Taq (Takara Bio Inc.) and gene-specific primers (S1 Table). The 18S rRNA was used as an internal control to normalize all data.

Fluorescence Microscopy

NO was visualized using the NO-specific fluorescent probe DAF-FM DA (Sigma-Aldrich), according to Wang’s method [70] with some modifications. Seven-day-old wild-type and mutant seedlings were incubated in 1 ml of 0.5× liquid MS medium (pH 5.8) with 10 μM DAF-FM DA for 20 min. Thereafter, the roots were washed three times for 15 min each in 0.5× liquid MS medium prior to visualization using a fluorescence microscope (ELLIPE TE2000-U; Nikon, Tokyo, Japan). The signal intensities were quantified using MetaMorph (Molecular Devices, Sunnyvale, CA).

Vector Construction and the Generation of Transgenic Plants

To detect the tissue-specific expression of GSNOR, an GSNORpro:GUS construct was generated by introducing the GSNOR promoter fragment (2.21 kb) in front of the GUS coding sequence in the PstI and XbaI sites of pCAMBIA1300. Detailed primer sequences are given in S1 Table. GUS staining assays were performed as described previously [71].

To determine the subcellular localization of GSNOR, GSNOR was engineered into pMDC83 with GFP at the C-terminus under the control of the 35S promoter. Detailed primer sequences are given in S1 Table. Transiently transfected tobacco leaves were imaged for GFP fluorescence using a Zeiss LSM710 confocal laser scanning microscope (Carl Zeiss AG, Jena, Germany).

For the overexpression of GSNOR, CaM1, and CaM4, the coding sequence of GSNOR was introduced into the SpeI and AscI sites of PMDC83 under the control of the 35S promoter, while the coding sequences of CaM1 and CaM4 were respectively introduced into the XbaI and BamHI sites of pCAMBIA1300 under the control of the 35S promoter. To generate constructs for the complementation of gsnor and cam1/4-1, a genomic DNA fragment of GSNOR was amplified and cloned into the HindIII and AscI sites of pMDC83, and a genomic DNA fragment of CaM4 was amplified and cloned into the SphI and XbaI sites of pCAMBIA1300. Arabidopsis transformation with Agrobacterium tumefaciens (strain GV3101) was performed by the floral dip method [72]. Homozygous T3 transgenic lines were used for further analysis. Detailed primer sequences are given in S1 Table.

BiFC Assays

BiFC assays were conducted as described previously [73]. Full-length GSNOR and CaM4 were cloned into either pSPYNE-35s or pSPYCE-35s. The resulting constructs were transiently expressed in 3-week-old tobacco leaves by Agrobacterium-mediated infiltration (strain GV3101). The YFP fluorescence of the tobacco leaves was imaged 2 days after infiltration using a Zeiss LSM710 confocal laser scanning microscope (Carl Zeiss AG).

Expression of Recombinant Proteins in E. coli and Overlay Assays

GST and recombinant GST-GSNOR, GST-GSNOR^N, GST-GSNOR^C, CaM4-GST, or CaM4-HiS were expressed in E. coli strain BL21. Expression of the GST fusion proteins was induced by treatment with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside for 4 h at 30°C; the proteins were purified as described previously [74], with some modifications.

In total, 1 μg of each purified GST fusion protein was separated by 10% SDS-PAGE and transferred to an Immobilon-P (polyvinylidene difluoride) membrane (Merck KGaA, Darmstadt, Germany), after which the expressed GST fusion proteins were detected using a monoclonal GST-specific antibody. To examine the CaM4 binding ability of the recombinant proteins, a duplicate blot was probed with 1 μg of CaM4-His (carrying a 6× histidine tag at the C-terminus) in the presence of 0.1 mM CaCl₂ or 0.1 EGTA in 10 ml of TBST and then probed with an anti-His monoclonal antibody (Proteintech Group Inc., Chicago, IL). Bound CaM4 was visualized using an ECL detection system (GE Healthcare, Wauwatosa, WI).

Protein Extraction, Immunoblotting, and In Vivo Co-IP

Arabidopsis proteins were extracted from 10-day-old seedlings using NEB buffer (20 mM HEPES, pH 7.5, containing 40 mM KCl, 1 mM EDTA, 1 mM PMSF, and 1× protease inhibitor cocktail [Roche, Basel, Switzerland]) after centrifugation at 12,000 × g, 4°C, for 20 min. Co-IP was performed as described previously [75,76], with minor modifications. In total, 50 μl of the supernatant was collected as input. The rest of the supernatant was used for immunoprecipitation using 10 μl of GFP-Trap agarose beads (ChromoTek, Martinsried, Germany). After incubation for 2 h at 4°C, the beads were washed five times in wash buffer (20 mM HEPES, pH 7.5, 40 mM KCl, and 0.1% Triton X-100). An appropriate amount of 2× SDS sample buffer was then added to the beads, which were boiled for 10 min at 100°C, and then subjected to 10% SDS-PAGE for immunoblot analysis using antibodies against GFP (G1544-100UG; Sigma-Aldrich) and GSNOR (AS09647; Agrisera, Vännäs, Sweden). All experiments were repeated independently three times; representative results from a single experiment are shown.

Measurement of GSNOR Activity

Total protein was extracted from the leaves of 7-d-old seedlings using NEB buffer (20 mM HEPES, pH 7.5, 40 mM KCl, and 1 mM EDTA) by centrifugation at 20,000g, 4°C for 20 min.

A total of each supernatant was incubated with 20 μl protein A beads (GE Healthcare) bound to GSNOR antibodies After 2 h, the beads were washed five times in wash buffer (20 mM HEPES, pH 7.5, 40 mM KCl, and 0.1% Triton X-100). An appropriate amount of 0.1M citric acid PH 3.0 was then added to elute the antibody. Immediately the eluted fractions were neutralize with 1M Tris-HCl, PH8.5-PH7.4 to obtain purified GSNOR protein.

GSNOR activity was measured by monitoring the decomposition of NADH [77,78]. The oxidation of NADH, dependent on the presence of the substrate GSNO, was determined spectrophotometrically at 340 nm. A crude leaf (25 mg) extract of Arabidopsis seedlings was prepared in 100 μl of 0.05 M HEPES buffer (20% glycerol, 10 mM MgCl₂, 1 mM EDTA, 1 mM benzamidine, and 1 mM Ɛ-aminocaproic acid, pH 8.0), centrifuged to remove insoluble material, and then clarified with a desalting column (Zeba desalting column; Pierce, Rockford, IL). Enzyme activity was determined at 25°C by incubating the desalted fraction (10 μl) in 180 μl of 0.1 M phosphate buffer containing 10 μl of 6 mM NADH as a cofactor and 10 ml of 6 mM GSNO as the substrate. GSNOR activity was monitored for 1 min after the addition of NADH using an Agilent 8453 UV spectrophotometer (Agilent Technologies, Santa Clara, CA). The rates were corrected for background NADH decomposition in each extract containing no GSNO. The rates were averaged over selected intervals during which the decrease in absorbance was linear. The final NADH decomposition values were normalized against the amount of total protein. All data given are the means of three independent experiments.

Determination of SNO Content

Total SNO levels were determined by Saville’s method [48,79]. Proteins were extracted in 100 mM Tris HCl, pH 6.8. The extracts were incubated for 5 min with an equivalent volume of solution A (1% sulfanilamide dissolved in 0.5 M HCl) in the presence or absence of solution B (solution A plus 0.2% HgCl₂), allowing the development of the diazonium salt. The formation of the azo dye product was obtained by reacting the two samples for an additional 5 min with an equal volume of solution C [0.02% of N-(1-naphthyl) ethylenediamine dihydrochloride dissolved in 0.5 M HCl], and the absorbance was subsequently read at 550 nm with a spectrophotometer. S-NOHCy was quantified as the difference of absorbance between solution B and A (B–A), comparing the values with a standard curve made from a solution of GSNO (Sigma-Aldrich). Low Mr SNOs were determined in the fraction passing through a 5 K cut of ultrafiltration membrane. The results were normalized against whole cell-lysate protein content.

Determination of Elemental Concentrations

To determine the Na⁺ and K⁺ contents in plant tissue, samples were harvested, oven-dried for at least 24 h at 80°C, weighed, and then digested in concentrated (69%, v/v) HNO₃ for at least 12 h for elemental extraction. The concentrations of Na⁺ and K⁺ were determined in appropriately diluted samples in an air-acetylene flame by atomic absorption spectrophotometry using a double-beam optical system with deuterium arc background correction (AAnalyst 100; PerkinElmer, Waltham, MA). Measurement of the Na⁺ and K⁺ concentrations was performed as described previously [80].

Accession Numbers

The sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL database under the following accession numbers: At5g43940 for GSNOR, At5g37780 for AtCaM1, At2g27030 for AtCaM2, At3g56800 for AtCaM3, At1g66410 for AtCaM4, At2g41110 for AtCaM5, At5g21274 for AtCaM6, and At3g43810 for AtCaM7.