Calcium dynamics and modulation in carrot somatic embryogenesis

Abstract

Free calcium (Ca²⁺) is a pivotal player in different in vivo and in vitro morphogenic processes. In the induction of somatic embryogenesis, its role has been demonstrated in different species. In carrot, however, this role has been more controversial. In this work, we developed carrot lines expressing cameleon Ca²⁺ sensors. With them, Ca²⁺ levels and distribution in the different embryogenic structures formed during the induction and development of somatic embryos were analyzed by FRET. We also used different chemicals to modulate intracellular Ca²⁺ levels (CaCl₂, ionophore A23187, EGTA), to inhibit calmodulin (W-7) and to inhibit callose synthesis (2-deoxy-D-glucose) at different times, principally during the first stages of embryo induction. Our results showed that high Ca²⁺ levels and the development of a callose layer are markers of cells induced to embryogenesis, which are the precursors of somatic embryos. Disorganized calli and embryogenic masses have different Ca²⁺ patterns associated to their embryogenic competence, with higher levels in embryogenic cells than in callus cells. The efficiency of somatic embryogenesis in carrot can be effectively modulated by allowing, within a range, more Ca²⁺ to enter the cell to act as a second messenger to trigger embryogenesis induction. Once induced, Ca²⁺-calmodulin signaling seems related with the transcriptional remodeling needed for embryo progression, and alterations of Ca²⁺ or calmodulin levels negatively affect the efficiency of the process.

Keywords: DAUCUS carota; EGTA; FRET; W-7; callose; in vitro culture; ionophore A23187; morphogenesis.

Figure 1

Carrot transformation and regeneration using protocol E. (A) Hypocotyl explants after 15 days in Selection 1 medium. (B) Hypocotyl explants after one month in Selection 2 medium. Arrows point to green, growing calli resistant to the selective agents. (C) Calli after one month in EIM with selective agents. (D) Full embryos developed in solid MS2% medium. Arrowheads point to developing somatic embryos. (E) Agarose electrophoresis gel showing PCR amplification of the transgene. From left to right, M, DNA molecular weight marker; R1-R8, Eight plantlets regenerated from the four resistant calli; WT, Wild type plant; P, PM-YC3.6-LTI6b plasmid; NC, PCR negative control. Bars: (A-C): 1 cm; D: 1 mm.

Figure 2

Carrot somatic embryogenesis. (A) Cell clumps after one week in liquid EIM. (B) Compact embryogenic masses after one week in liquid MS2%. (C-F) Time-lapse images of an immobilized culture one week (C), two weeks (D), three weeks (E) and one month after immobilization (F). Arrowheads point to individual developing embryos progressing through different developmental stages. Arrows point to an individual proliferating callus mass. (G) Fully developed embryo after three weeks in liquid MS2%. (H) Embryos germinated in solid MS2%. (I) Acclimatized adult carrot plant regenerated from somatic embryos. Bars: (A, B): 50 µm; C-H: 500 µm.

Figure 3

FRET imaging of Ca²⁺ levels during carrot somatic embryogenesis in PM-YC3.6-LTI6b cameleon lines. Each pair of images shows the same field imaged by phase contrast (left) and FRET ratio (YFP/CFP emissions; right). The LUT bar displays the false coloration of FRET ratios. (A) One-week-old cell clumps. Note the higher Ca²⁺ levels of the cells at the left side (arrowheads). (B) Cell clump transforming into an embryogenic mass. Note the higher Ca²⁺ levels of the cell mass at the left side (arrowhead). (C, D) Two-weeks-old compact, globular embryo (C) and callus mass (D). Arrowhead in C points to a pole with higher Ca²⁺ levels. (E) Three-weeks-old heart-shaped embryo. Arrowheads point to the cotyledon primordia. (F) Torpedo embryo. (G, H) Shoot (G) and root (H) apical regions of a cotyledonary embryo. Bars: (A-E, G, H): 40 µm; F: 100 µm.

Figure 4

Callose deposition in cultured embryogenic structures. Each pair of (A-E) images shows the same field of aniline blue-stained samples imaged by phase contrast (left) and fluorescence (right). (A, B) Small clusters of callose-positive cells in cell clumps (A) and proembryogenic masses (B). Note the intense aniline blue staining in the cell wall region of specific cells (arrowheads). (C) Irregular callus mass with loosely connected cells. (D, E) Small cell clump (D) and proembryogenic mass (E) from cultures with 5 mM 2-deoxy-D-glucose. (F) Modulation of callose deposition by the addition of 2-deoxy-D-glucose at different concentrations and 3-day, 7-day and continuous exposures. The effects of the treatments are expressed as percentages of embryos produced (% embryos). Letters represent significant differences with respect to their respective controls according to the LSD test. Bars: (A, B, D, E): 40 µm; C: 80 µm.

Figure 5

Modulation of intracellular Ca²⁺ homeostasis by the addition of CaCl₂ (A), the Ca²⁺ channel ionophore A23187 (B), EGTA, a Ca²⁺ chelator (C), and W-7, a CaM antagonist (D) at different concentrations and 3-day, 7-day and continuous exposures. The effects of the treatments are expressed as percentages of embryos produced (% embryos) ± standard error. At least three different repeats were performed for each chemical, concentration and exposure time applied. Data were analyzed using the ANOVA test and groups of significance were established according to the least significant difference (LSD) test (p ≤ 0.05). Different letters represent significant differences with respect to their respective controls according to the LSD test.

Figure 6

Carrot somatic embryos produced in cultures with added CaCl₂ (A-C), ionophore A23187 (D-F), EGTA (G-I) and (J-L) at different concentrations and durations, as described in the images. Arrows point to disorganized callus masses. Bars: 1 mm.