CML24, regulated in expression by diverse stimuli, encodes a potential Ca2+ sensor that functions in responses to abscisic acid, daylength, and ion stress

Abstract

Changes in intracellular calcium (Ca(2+)) levels serve to signal responses to diverse stimuli. Ca(2+) signals are likely perceived through proteins that bind Ca(2+), undergo conformation changes following Ca(2+) binding, and interact with target proteins. The 50-member calmodulin-like (CML) Arabidopsis (Arabidopsis thaliana) family encodes proteins containing the predicted Ca(2+)-binding EF-hand motif. The functions of virtually all these proteins are unknown. CML24, also known as TCH2, shares over 40% amino acid sequence identity with calmodulin, has four EF hands, and undergoes Ca(2+)-dependent changes in hydrophobic interaction chromatography and migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca(2+) and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs, and transcript levels are increased from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA), and indole-3-acetic acid. However, CML24 protein accumulation changes were not detectable. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress; in regions undergoing growth; in vascular tissues and various floral organs; and in stomata, trichomes, and hydathodes. CML24-underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, are defective in long-day induction of flowering, and have enhanced tolerance to CoCl(2), molybdic acid, ZnSO(4), and MgCl(2). MgCl(2) tolerance is not due to reduced uptake or to elevated Ca(2+) accumulation. Together, these data present evidence that CML24, a gene expressed in diverse organs and responsive to diverse stimuli, encodes a potential Ca(2+) sensor that may function to enable responses to ABA, daylength, and presence of various salts.

Figure 1.

CML24 encodes a CaM-like, Ca²⁺-binding protein. A, Amino acid sequence of CML24 and the four Arabidopsis CaM isoforms represented by CaM1, CaM2, CaM6, and CaM7. Shaded and boxed residues indicate similarity and identity, respectively. The Ca²⁺-binding domains of the four EF hands are underlined. B. Western blot analyzed with anti-CML24 antibody. The faster migration of CML24 through the SDS-polyacrylamide gel after incubation with Ca²⁺ is an indication that CML24 can bind Ca²⁺ and change conformation upon interaction with Ca²⁺.

Figure 2.

CML24 and CML24::GUS expression. A, CML24 expression is induced by a variety of stimuli. Plants were left alone (controls; black bars) or treated (white bars) with the following stimuli and harvested at the time points indicated in parentheses: touch (30 min), dark (30 min), 37°C heat (1 h), 4°C cold (4 h), 20 mm H₂O₂ (30 min), 100 μm ABA (30 min), 100 μm IAA (30 min), 100 μm ACC (45 min), and 100 μm GA (45 min). QRT-PCR was conducted as described in “Materials and Methods”; three independent biological replicates were analyzed. Error bars represent se. B, CML24 is expressed in many plant organs. Total RNA purified from roots of 4-week-old plants (Rt), and rosette leaves (Ros), inflorescence stems (St), cauline leaves (Cau), flowers (Fl), and siliques (Sil) of 7-week-old plants was subjected to RT-PCR with primers specific for CML24 and TUB4 (encoding tubulin). TUB4 serves as a control for RNA integrity and as a semiquantitative standard. C to P, CML24::GUS is expressed in many plant organs and throughout plant development. CML24::GUS is expressed in (C) the shed seed coat, (D) 2-d-old seedlings, (E) 7-d-old seedlings, (F) guard cells, (G) leaf vasculature and trichomes of 14-d-old seedlings, (H) 6-d-old dark-grown seedlings, (I) hydathodes, (J) branch points, (K) developing seed and abscission zone, (L) styles of young flowers, (M) mature anthers and stigmatic papillae, (N) root vasculature and lateral root initiation sites (arrow) of 7-d-old seedlings, and (O) lateral root tip and (P) primary root tip of 14-d-old seedlings. Bars = 100 μm in C, D, G, H, and K to P; bars = 1 mm in E and I; bars = 5 μm in F; bars = 500 μm in J.

Figure 3.

CML24-underexpressing transgenics (U1, U2) have reduced CML24 levels, and this effect is likely gene specific. A, Western blot of total protein from wild-type (WT) and transgenic plants interacted with anti-CML24 antibodies. The antibody cross reacts with an abundant, larger protein that serves as a loading control. U1 and U2 have undetectable levels of CML24. Lanes O1 and O2 serve to verify the migration position of CML24 as these samples are from transgenics engineered to overexpress CML24. B, Semiquantitative RT-PCR of CML23 and TUB4 (control) from wild type, U1, and U2. PCR products after cycles 28 and 34 were analyzed by gel electrophoresis. No effect on CML23 expression is detected in the CML24-underexpressing transgenics.

Figure 4.

CML24-underexpressing transgenics (U1, U2) show altered responses to ABA. U1 and U2 are less sensitive to ABA-induced dormancy. The graph to the left shows a greater percentage of U1 and U2 seedlings germinate on 1 μm and 2 μm ABA 6 d post sowing than wild type (WT). n ≥ 80 on PN and n ≥ 140 on ABA. The photograph on the right shows U1 and U2 seedlings have more enhanced development and are greener than wild type when grown on media supplemented with 5 μm ABA and 0.5% Suc. Scale bar = 15 mm.

Figure 5.

CML24-underexpressing transgenics (U1, U2) flower late in long days. A, Seven-week-old wild-type plants flower earlier than U1 and U2 when grown in a 24-h photoperiod. B, Rosette leaf numbers for plants grown in a 16-h photoperiod (LD, n ≥ 13), an 8-h photoperiod (sd, n ≥ 10), after a 40-d vernalization treatment (Vern, n ≥ 14), or in response to a 100 μm GA treatment (GA, n ≥ 33). Error bars represent sds. Flowering time was recorded for plants grown in (C) a 16-h photoperiod, n ≥ 13; (D) an 8-h photoperiod, n ≥ 10; (E) after a 40-d vernalization treatment, n ≥ 14; and (F) in response to a 100-μm GA treatment, n ≥ 33.

Figure 6.

The growth of the CML24-underexpressing transgenics (U1, U2) is more tolerant of CoCl₂, molybdic acid, and ZnSO₄ than that of wild type. A, Representative wild type (WT, left) and CML24-underexpressing transgenic (U1, right) 20 d after growth on PN media supplemented with 100 μm CoCl₂. Wild type is chlorotic and does not develop a first set of true leaves. The CML24-underexpressing transgenics maintain some chlorophyll in the cotyledons and have expanded true leaves. B, U1 and U2 produce more chlorophyll (Chl) per seedling than wild type when grown on 100 μm CoCl₂. The average chlorophyll per seedling was determined by dividing the total chlorophyll absorbance (A₆₅₂) by the total number of seedlings in a pool. The number of seedling per pool was ≥10; three pools were analyzed. C, Wild type, U1, and U2 have comparable chlorophyll levels when grown on unsupplemented PN growth media; n ≥ 5. D, Representative 4-d-old wild type (left) and CML24-underexpressing transgenic (right) plant grown on PNS PN supplemented with of 2 mm molybdic acid (Na₂MoO₄). E, U1 and U2 develop expanded cotyledons at a faster rate than wild type; n ≥ 10. F, Seedlings grown vertically on PN supplemented with 250 μm ZnSO₄ (top) or unsupplemented PN (bottom) for 10 d. G, U1 and U2 develop longer roots than wild type after 10-d growth on 250 μm ZnSO4; n ≥ 39. H, Wild type, U1, and U2 have comparable roots lengths on unsupplemented PN; n ≥ 29. Error bars represent sds. Scale bars = 1 mm in A and D, and 10 mm in F.

Figure 7.

The CML24-underexpressing transgenics (U1, U2) are more tolerant of MgCl₂ than wild type (WT) and accumulate comparable levels of Mg²⁺ and Ca²⁺. A, U1 and U2 show further development than wild type on growth media supplemented 25 mm MgCl₂ (left) or 30 mm MgCl₂ (right) for 20 d. Scale bar = 15 μm. B, Chlorophyll (Chl) levels were determined on increasing concentrations of MgCl₂ to represent differences in vegetative development. Error bars represent sds, n ≥ 10. C and D, ICP-MS was performed on approximately 3-week-old plants grown on increasing concentrations of MgCl₂. C, Wild type, U1, and U2 accumulate comparably increased levels of Mg²⁺ when grown on PNS supplemented with increasing concentration of MgCl₂. D, All plants accumulate reduced levels of Ca²⁺ when grown on increasing concentrations of MgCl₂. Error bars represent sds of four pools of four seedlings per pool for 0 to 15 mm MgCl₂ or three pools of two to five seedlings per pool for 25 and 35 mm MgCl_2. PPM, Parts per million.