Arabidopsis potential calcium sensors regulate nitric oxide levels and the transition to flowering

Abstract

In plants, flowering is a critical developmental transition orchestrated by four regulatory pathways. Distinct alleles encoding mutant forms of the Arabidopsis potential calcium sensor CML24 cause alterations in flowering time. CML24 can act as a switch in the response to day length perception; loss-of-function cml24 mutants are late flowering under long days, whereas apparent gain of CML24 function results in early flowering. CML24 function is required for proper CONSTANS (CO) expression; components upstream of CO in the photoperiod pathway are largely unaffected in the cml24 mutants. In conjunction with CML23, a related calmodulin-like protein, CML24 also inhibits FLOWERING LOCUS C (FLC) expression and therefore impacts the autonomous regulatory pathway of the transition to flowering. Nitric oxide (NO) levels are elevated in cml23/cml24 double mutants and are largely responsible for FLC transcript accumulation. Therefore, CML23 and CML24 are potential calcium sensors that have partially overlapping function that may act to transduce calcium signals to regulate NO accumulation. In turn, NO levels influence the transition to flowering through both the photoperiod and autonomous regulatory pathways.

Keywords: calcium; calmodulin; cell signaling; flowering; nitric oxide.

Figure 1

CML23 and CML24 are calmodulin-like proteins. Amino acid sequence alignment of Arabidopsis CML24 (At5g37770), CML23 (At1g66400), and one of the calmodulins, CaM2 (At2g41110). Shaded amino acids indicate sequence similarity, underlines delineate EF-hand Ca²⁺ binding loops, letters above the CML24 sequence indicate amino acid substitutions in the cml24-1, cml24-2, and cml24-4 mutants, the solid triangles indicate the two independent insertion sites of the T-DNA insertions in CML23. Amino acid numbers are at right.

Figure 2

CML23:GUS is expressed in many organs and throughout development. CML23:GUS is expressed in (A) four-day-old seedlings, (B) cauline leaves, (C) guard cells, (D) branch junctions on the inflorescence stem, (E) root tip, (F) sites of lateral root formation, (G) flower organs, (H) funiculi and (I) siliques.

Figure 3

Mutant CML24 proteins show a Ca²⁺-dependent mobility shift. Wild type (Col-0) and mutant CML24 proteins (cml24-1, cml24-2 and cml24-4) were analyzed along side purified CML24 produced heterologously in E. coli (CML24_rec). Protein samples contained either 5 mM CaCl₂ (+) or 5 mM EGTA (−) and were separated by 13% SDS-PAGE. The proteins were transferred to nitrocellulose membrane and probed with anti-CML24 antibody. The faster migration of wild-type, mutant, and recombinant CML24 in the presence of Ca²⁺ than in the presence of EGTA indicates that mutations in cml24-1, cml24-2 and cml24-4 do not completely impair Ca²⁺ binding and consequent conformational changes.

Figure 4

CML23 and CML24 are required for appropriate timing of the transition to flowering. Wild type (Col-0) and mutants were grown in16-hour photoperiods (A) or short-day eight-hour (B) and seven-hour (C) photoperiods and the percentages of plants flowering over time were recorded. Rosette leaf numbers at flowering onset from (A–C) were recorded for Col-0 and mutants grown under (D) long-day and (E) short-day photoperiods. Values are means ± SEM (n = 6 to 33).

Figure 5

Regulation of flowering time gene expression in cml mutants. Wild type and cml mutants were grown for two weeks in 16-hour photoperiods and harvested at four-hour intervals over a 24-hour period (A, D, E and F) or 11 hours after dawn (A–C). Quantitative RT-PCR was performed to detect abundances of (A) FT, (B) SOC1, (C) FLC, (D) CO, (E) GI, (F) FKF1 transcripts relative to TUB4 (encoding tubulin) transcripts. In (A), Col-0 represented by the solid bar; cml24-2 is the open bar. The light and dark periods for (A) and (D–F) are represented by the open bars and filled bar, respectively, above (A and D). Values are means ± SEM (n = 3 to 5).

Figure 6

CML23 and CML24 regulate NO accumulation in leaves. (A) Rosette leaves of two-week-old Col-0 and cml mutants were treated with or without 0.4 mM cPTIO (NO scavenger) for four hours and then stained with DAF-FM DA (NO sensitive fluorescence dye). Fluorescence was detected with 490–495 nm excitation and 515 nm emission. (B) Average fluorescence intensity levels from the rosette leaves corresponding to the treatments in (A) were quantified using Image J (NIH). Values are means ± SEM (n = 6).

Figure 7

Altered NO levels affect FLC transcript levels. Quantitative RT-PCR of FLC levels relative to TUB4 were determined in 2-week-old plants grown under long-day (16-hr) photoperiods and treated with or without 0.4 mM cPTIO for 4 hours. Values are means ± SEM (n = 3 to 4).