INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

In plants Ca²⁺ ion acts as a second messenger in the signal transduction of a variety of environmental stimuli (Bush, 1995). In analogy to animal systems, stimulus-induced elevations in cytosolic Ca²⁺ are believed to be perceived and transduced by Ca²⁺-binding proteins and protein kinases (Poovaiah and Reddy, 1993; Trewavas, 1997).

The environmental stimuli and signaling events that trigger and regulate plant embryogenesis are largely unknown. The small size and relative inaccessibility of zygotic embryos in the seeds have contributed to the lack of understanding of this developmental process. Somatic embryogenesis, nevertheless, serves as an alternative experimental system (Zimmerman, 1993). Work on the carrot system has shown that Ca²⁺ enhances embryogenic frequency (Jansen et al., 1990), and its deprivation arrests somatic embryo formation (Overvoorde and Grimes, 1994). These observations are suggestive of an intermediary role for Ca²⁺ during plant embryogenesis.

The role of calmodulin (CaM) as a Ca²⁺ modulator during somatic embryogenesis has also been investigated (Overvoorde and Grimes, 1994). Although, Ca²⁺-CaM complexes have been localized in the actively dividing regions of carrot somatic embryos, its role as the Ca²⁺ modulator regulating the process of embryogenesis has not been ascertained. Furthermore it is now well established that plants predominantly exhibit a Ca²⁺-dependent, CaM-independent protein kinase activity. The enzymes responsible for such an activity, namely Ca²⁺-dependent protein kinases (CDPKs), are encoded by a multigene family and are ubiquitous in plants (Roberts, 1993). CDPKs are unique in having a C-terminal CaM-like domain that can directly bind Ca²⁺, thereby making CaM unnecessary for their activation (Harper et al., 1991). CDPKs are now implicated to play an intermediary and regulatory role in a variety of developmental and metabolic processes (McMichael et al., 1995; Huber et al., 1996; MacIntosh et al., 1996). We have earlier reported a 55-kD soluble sandalwood (Santalum album) Ca²⁺-dependent protein kinase (swCDPK) that accumulates only in somatic/zygotic embryos, endosperm, and seedlings (Anil et al., 2000). The developmentally regulated, tissue-specific expression of this CDPK isoform in sandalwood suggests its involvement in early developmental processes such as embryogenesis, seed development, and germination.

In the present study we have investigated the role of Ca²⁺ as a second messenger and examined the probable involvement of Ca²⁺-mediated signaling pathway(s) in the induction/regulation of somatic embryogenesis in sandalwood. Changes in [Ca²⁺]_cyt of pro-embryogenic cell masses (PEMs) were monitored when exposed to culture conditions conducive for embryo differentiation. Increase in [Ca²⁺]_cyt was recorded in PEMs upon their transfer from the callus proliferation medium that contains 2,4-dichlorophen-oxyacetic acid (2,4-D) to the embryo differentiation medium that lacks 2,4-D and contains higher osmoticum. The study identifies exogenous Ca²⁺ as the source for such an influx and examines the necessity of this Ca²⁺ pool for the process of embryo differentiation. The necessity of exogenous Ca²⁺ pool for the accumulation and activity of swCDPK in embryogenic cultures has also been investigated.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Sandalwood Somatic Embryogenesis

Endosperm of sandalwood dedifferentiated into callus in Murashige and Skoog (MS) medium containing benzylaminopurine and 2,4-D (MS₁). These cells proliferated into a friable embryogenic callus in the presence of 2,4-D alone in the medium (MS₂). The embryogenic callus consisted of two cell types: small, richly cytoplasmic embryogenic cells that aggregate into clumps (PEMs) and the larger elongated nonembryogenic cells containing scanty cytoplasm and large vacuole. MS₃ medium that lacks 2,4-D and contains 2% (w/v) mannitol (differentiation medium)-induced development of somatic embryos from the PEMs. However, under similar growth conditions nonembryogenic cells fail to differentiate into embryos.

Incorporation of ⁴⁵Ca²⁺ and Measurement of Cytosolic Ca²⁺ in the Embryogenic Cells

A 4-fold higher ⁴⁵Ca²⁺ uptake was observed in PEMs that were incubated in MS₃ as compared with those retained in the callus proliferation medium (Fig. 1, A and B). PEMs washed with Triton X-100 overnight showed only 15% of ⁴⁵Ca²⁺ incorporation. This represented cell wall sequestered ⁴⁵Ca²⁺ and has been subtracted from the uptake observed without the detergent wash. Therefore, the assay reflects the ⁴⁵Ca²⁺ incorporated into the symplast.

View larger version (20K): [in this window] [in a new window]   Figure 1.   Incorporation of 45Ca2+ into symplast of PEMs. A, Graph represents the percentage incorporation of 45Ca2+ in callus proliferation medium (MS2), embryo differentiation medium (MS3), and in embryo differentiation medium + 1 mM EGTA (MS3 + EGTA). 100% represents 10,389 cpm obtained from 0.02 µCi of 45Ca2+ added per reaction. B, Quantity of 45Ca2+ incorporated into PEMs suspended in MS2, MS3, or MS3 + EGTA. B represents mean values obtained from 10 determinations.

For the purpose of cytosolic Ca²⁺ measurements, loading of fura-2AM (AM is a commerical notation to denote the ester linkage to fura-2) into PEMs was efficient when incubated for 2 h with 0.02% (v/v) pluronic-127. The Ca²⁺-bound-dye fluorescence obtained by excitation at 351 nm was negligible in PEMs maintained in callus proliferation medium (data not shown). This fluorescence became intense when the PEMs were perfused with embryo differentiation medium (Fig. 2, A and A'). However, nonembryogenic cells failed to show a similar increase in Ca²⁺-bound dye fluorescence, upon their transfer to the differentiation medium (Fig. 2, B and B'). Confocal-time-based series of images clearly showed an increase in the Ca²⁺-bound dye fluorescence upon transfer of PEMs from the callus proliferation medium to the embryo differentiation medium (Fig. 3). Ratiometric analysis of fura-2 fluorescence (351-/361-nm excitation) from the confocal-images showed increase in the [Ca²⁺]_cyt in PEMs, from a "resting" concentration of 30 to 50 nM in MS₂ to a concentration of 650 to 800 nM (n = 10 cells) when perfused with MS₃ medium (Fig. 4A). This influx was not observed when perfused with MS₃ containing 1 mM EGTA (Fig. 4B), thus indicating that Ca²⁺ influx must originate from an exogenous pool and enter the cytosol via the plasma membrane.

View larger version (98K): [in this window] [in a new window]   Figure 2.   Ca2+-Bound fura-2 fluorescence obtained with excitation wavelength of 351 nm. A, Bright field image of fura-2 loaded pro-embryogenic cells; A', fluorescence image obtained upon transfer of the PEM from MS2 to MS3 medium; B, bright field image of fura loaded nonembryogenic cells; B', fluorescence image after transfer of the nonembryogenic cells from MS2 to MS3 medium. Scale bars = 15 µm.

View larger version (24K): [in this window] [in a new window]   Figure 3.   Confocal time-based series of images showing Ca2+ homeostasis when fura-2 loaded PEMs in MS2 (MS + 1 mg/L 2,4-D) are perfused with MS3 (MS + 2% [v/v] mannitol). A through I represent the free-dye fluorescence obtained by excitation at 361 nm from 0 time point of perfusion with MS3; A' through I' represent the Ca2+-bound dye fluorescence obtained by excitation at 351 nm at identical time points. Negligible fluorescence in A' through C' indicates the low resting [Ca2+]cyt in pro-embryogenic cells for few seconds after perfusion with MS3 medium. Intense fluorescence in D' represents an influx of Ca2+ into the cytosol, which gradually decreases with time (E'-I'). Scale bars = 15 µm.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Ca2+ homeostasis in the cytosol of PEMs. Fura-2-loaded pro-embryogenic cells were first perfused with MS2 (MS + 1 mg/L 2,4-D) to establish the resting [Ca2+]cyt. The cells were challenged (arrow) with: A, MS3 (MS + 2% [w/v] mannitol); and B, MS3 + 1 mM EGTA. Confocal time-based series of images that depict time-based alterations in Ca2+-bound dye fluorescence and free dye fluorescence in PEMs have been used to determine changes in [Ca2+]cyt by the double wave length ratiometric analysis.

Effect of Ca²⁺ Chelation, Ca²⁺-Channel Blockers, A23187, and N-(6-Aminohexyl)-5-Chloro-1-Naphthalene Sulfonamide Treatments on Embryogenesis

Under optimal differentiation conditions, each PEM gave rise to a cluster of six to eight bipolar embryos. By d 21 of the differentiation cycle, the culture predominantly contained a mixture of bipolar- and torpedo-stage embryos. The dimethyl sulfoxide (DMSO)-treated embryogenic clumps showed no perceivable variation in embryogenic frequency as compared with those developed under optimal differentiation conditions and therefore were considered to exhibit 100% embryogenesis. The percentage embryogenesis obtained with various Ca²⁺ antagonist treatments have been calculated relative to the controls mentioned.

Embryogenesis was completely arrested when all residual Ca²⁺ was chelated from the embryo differentiation medium with the addition of 1 mM EGTA (Fig. 5A). Increasing the concentration of plasma membrane Ca²⁺-channel blockers, namely bepridil, nifedipine, and verapamil, in the differentiation medium also reduced the frequency of embryogenesis (Fig. 5B), and at concentrations of 500 µM either nifedipine or bepridil, a complete arrest of embryo development was observed (Fig. 5B). Verapamil at 500 µM reduced embryogenesis by 65% compared with the control (Fig. 5B). Differentiation medium containing 100 µM N-(6-aminohexyl)-5-chloro-1-na-phthalene sulfonamide (W7) or A23187 resulted in 85% and 64% reduction in embryogenesis, respectively (Fig. 5, C and D).

View larger version (26K): [in this window] [in a new window]   Figure 5.   Embryogenic frequency as a function of Ca2+/pharmacological agents in MS3. A, Percentage embryogenesis as a function of Ca2+ concentration in the differentiation medium; 0 mM Ca2+ was obtained by the addition of 1 mM EGTA into MS3 lacking CaCl2. B, Percentage embryogenesis as a function of increasing concentrations of the Ca2+-channel blockers, namely nifedipine, bepridil, and verapamil. C, Percentage embryogenesis as a function of increasing concentrations of W7. D, Percentage embryogenesis obtained with increasing concentration of the Ca2+ ionophore A23187. Percentage embryogenesis in the different culture treatments was calculated relative to the 100% embryogenesis obtained in unmodified MS3 or the DMSO control both containing 3.9 mM Ca2+.

PEMs grew normally and formed bipolar- and torpedo-stage embryos by d 21 of culture incubation in MS₃ medium containing DMSO (Fig. 6A). Although EGTA and Ca²⁺-channel blocker treatments resulted in arrest of embryo development, the constituent cells of PEMs continued to proliferate to form larger clumps, suggesting that arrest of embryogenesis was not a result of cell death (Fig. 6, B and C). Embryos formed were abnormally large in size and fused in nature in the case of cultures grown under A23187 or W7 treatments (Fig. 6, D and E).

View larger version (137K): [in this window] [in a new window]   Figure 6.   Embryo development under different culture treatments. A, Control showing bipolar- and torpedo-stage embryos emerging from embryogenic cell clumps after d 21 culture incubation; inset a, PEM as seen in the initial inoculum; B, enlarged cell clumps grown in MS3 medium containing 1 mM EGTA; C, enlarged cell clumps obtained in MS3 medium containing 500 µM nifedipine; D, fused embryos under Ca2+ ionophore treatment; E, enlarged and fused embryos under W7 treatment. Scale bar = 15 µm (inset a); = 6 mm (A-C), = 2 mm (D and E).

Bipolar embryos grown under optimal conditions showed distinct root and shoot apices and vascular procambial strands (Fig. 7A). The enlarged clumps obtained under Ca²⁺ chelated conditions had normal, compactly arranged cells (Fig. 7B). PEMs of the initial inoculum comprising of 20 to 50 cells per PEM showed a 10- to 100-fold increase in the number of cells by d 21 of culture incubation. No trace of any vascular development was observed in these enlarged cell clumps (Fig. 7B).

View larger version (100K): [in this window] [in a new window]   Figure 7.   Histological differences in the embryogenic cultures grown in the presence or the absence of Ca2+ in MS3. A, Longitudinal section of torpedo-stage embryo grown under optimal differentiation conditions showing distinct radical and plumular ends and vascular procambial strands. B, Section of an enlarged clump from cultures grown under Ca2+-chelated conditions showing compactly arranged cells but no trace of vascular development. Scale bar = 150 µm.

Protein Synthesis in Enlarged Embryogenic Clumps Grown in Ca²⁺-Chelated Differentiation Medium

Many of the soluble proteins that were present in cultures grown under optimal conditions were not detectable in embryogenic cultures grown under Ca²⁺-deprived/chelated conditions or A23187 culture treatments (Fig. 8A). Nevertheless, L-[³⁵S]Met labeling of proteins in cultures grown under Ca²⁺-chelated differentiation conditions (MS₃ + 1 mM EGTA) showed that protein synthesis was taking place in the enlarged cell clumps even at d 21 of culture incubation (Fig. 8B).

View larger version (51K): [in this window] [in a new window]   Figure 8.   Soluble proteins and in vivo protein synthesis under different culture treatments: A, 10% (w/v) SDS-polyacrylamide gel showing Coomassie-stained proteins. Arrows indicate prominent protein bands absent under A23187 treatment and Ca2+-chelated/deprived conditions as compared with the control; B, L-[35S]Met labeling of proteins in embryogenic cultures grown under optimal (control) and under Ca2+-chelated (EGTA) differentiation conditions.

swCDPK Accumulation and Activity under Different Culture Treatments

Changes in Ca²⁺-dependent substrate phosphorylation activity in the soluble protein extracts from different culture treatments are depicted in Figure 9A. Cultures under optimal differentiation conditions showed a 6.8-fold increase in the Ca²⁺-dependent phosphorylation of the in vitro substrate histone III-S. The W7-culture treatment at a concentration of 100 µM gave moderate Ca²⁺-dependent histone phosphorylation activity showing a 5.6-fold increase with addition of Ca²⁺. However, culture treatment with 1 mM EGTA and 100 µM Ca²⁺ ionophore resulted in low Ca²⁺-dependent histone phosphorylation activity, increasing only 2.6- and 1.25-fold, respectively, with addition of Ca²⁺. Cultures treated with 500 µM Ca²⁺-channel blocker did not exhibit any Ca²⁺-dependent histone kinase activity (Fig. 9A).

View larger version (28K): [in this window] [in a new window]   Figure 9.   CDPK activity and swCDPK protein in soluble protein extracts of embryogenic cultures subjected to treatments with different pharmacological agents. A, Ca2+-Dependent histone phosphorylation activity in embryogenic cultures grown under optimal differentiation conditions (Con), Ca2+-chelated conditions (EGTA), 100 µM Ca2+ ionophore (I), 500 µM nifedipine (Nif), 500 µM verapamil (Verp), and 500 µM bepridil (Bep) treatments. B, Ca2+-Dependent phosphorylation of the 55-kD swCDPK protein determined by assaying in the absence of exogenous substrate followed by SDS-PAGE. C, Immunodetection of the 55-kD swCDPK band using polyclonal antisoybean CDPK in soluble proteins from cultures grown under optimal conditions and different pharmacological treatments.

We have identified earlier a 55-kD CDPK in sandalwood embryogenic cultures that exhibited Ca²⁺-dependent autophosphorylation activity (Anil et al., 2000). W7 treatment of embryogenic cultures did not produce appreciable variation in the autophosphorylation activity of swCDPK relative to control cultures grown under optimal differentiation conditions (Fig. 9B). However, negligible phosphorylation of swCDPK was observed in proteins from cultures subjected to Ca²⁺ chelation or A23187 treatments (Fig. 9B) and the activity was undetectable in those cultures treated with 500 µM Ca²⁺ channel blocker (Fig. 9B). In corroboration the 55-kD swCDPK was strongly immunodetected in the soluble proteins of embryogenic cultures grown under optimal conditions and in cultures treated with W7 (Fig. 9C). It was not immunodetected in cultures grown under Ca²⁺-chelated, Ca²⁺-channel blocker, and Ca²⁺-ionophore culture treatments (Fig. 9C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The data presented in this paper show that the withdrawal of 2,4-D in conjunction with increased osmoticum in the medium can provoke a cytosolic elevation of Ca²⁺ in sandalwood pro-embryogenic cells. As it is the case with several other in vitro embryogenic systems (Komamine et al., 1992; Sankara Rao, 1996), sandalwood also requires the auxin 2,4-D for tissue dedifferentiation and for subsequent induction of embryogenic-competence in certain cells (PEMs) and withdrawal of 2,4-D for embryo differentiation from the PEMs. Mannitol in the differentiation medium (MS₃) helps simulate the desiccation experienced by seed embryos during maturation and onset of dormancy and facilitates proper embryo maturation, particularly in tree species (Merkle, 1995). Therefore this paper, in essence, provides evidence for cytosolic elevation of Ca²⁺ in pro-embryogenic cells of sandalwood when exposed to culture conditions conducive for embryogenic development.

Although, the ⁴⁵Ca²⁺-uptake studies suggest increased movement of exogenous Ca²⁺ into PEMs in the differentiation medium, it is possible that given conditions for differentiation, PEMs exhibit a non-specific increase in the uptake of nutrients and ions. However, fura-2-based ratiometric measurements of [Ca²⁺]_cyt confirmed the occurrence of cytosolic Ca²⁺ elevation in the PEMs when exposed to differentiation conditions. The arrest of such an influx by EGTA supports the observation that differentiation conditions induce movement of exogenous Ca²⁺ into the cytosol. Further, the absence of Ca²⁺ elevations in nonembryogenic cells exposed to identical culture conditions indicate the specificity of such a phenomenon to cells predisposed to develop into embryos.

The fold increase in Ca²⁺ uptake caused by transfer of PEMs from MS₂ to MS₃ was lower in determinations obtained with ⁴⁵Ca²⁺-incorporation studies as compared with that with fura-2-based measurement of [Ca²⁺]_cyt. This indicates that PEMs in the callus proliferation medium do also take up Ca²⁺ from the exogenous pool. However, the Ca²⁺ that is taken up must rapidly be sequestered into internal organelles so as to maintain the resting [Ca²⁺]_cyt of PEMs in the callus proliferation medium.

To validate the role of exogenous Ca²⁺ as a second messenger in the induction/regulation of somatic embryogenesis, the study investigates the necessity of this Ca²⁺ pool for somatic embryo development per se. The arrest of sandalwood somatic embryogenesis under Ca²⁺-chelated culture conditions (MS₃ + 1 mM EGTA) suggests that exogenous Ca²⁺ indeed is required for embryogenic development. Concomitantly, the inhibition of embryogenesis by plasma membrane Ca²⁺-channel blockers confirmed the necessity of this Ca²⁺ pool for the process of embryogenesis. However, it is interesting that PEMs continue to proliferate under such culture conditions, wherein exogenous Ca²⁺ has been chelated or deprived, exhibiting protein synthesis even at d 21 of culture incubation. This implies that although internally stored Ca²⁺ can sustain cell proliferation, it remains an inaccessible store for the process of embryo differentiation.

The Ca²⁺ ionophore A23187 allows diffusion of Ca²⁺ through membranes in an electrically neutral manner, thus neutralizing Ca²⁺ gradients that are normally present across the plasma membrane. Since Ca²⁺ ionophores create an artificial influx of Ca²⁺ into the cytoplasm, they can elicit Ca²⁺-mediated physiological responses even in the absence of an environmental stimulus (Poovaiah and Reddy, 1993). However, A23187 treatment in our study showed abnormal and reduced embryogenesis. A23187 culture treatment results in PEMs being exposed to extended periods of increased [Ca²⁺]_cyt. This could minimize the significance of normal transient Ca²⁺ influxes that occur specifically in response to a stimulus, thus interfering with the process of proper pattern formation and embryo differentiation.

The reduction in the percentage embryogenesis by W7 culture treatment indicates that CaM or a related Ca²⁺-modulated protein is involved in perceiving the Ca²⁺ signal. This gives strength to the presumption that somatic embryogenesis is regulated by Ca²⁺-mediated signaling pathway(s). In carrot somatic embryos activated CaM has been localized to regions undergoing rapid cell division, and increase in the level of CaM mRNA observed during globular- and heart-shaped stages (Overvoorde and Grimes, 1994). Yet the modulator role of CaM in regulation of embryogenesis remains unclear. Furthermore, sandalwood embryogenic cultures exhibit a Ca²⁺-dependent and CaM-independent protein kinase activity and accumulate a developmentally regulated, tissue-specific soluble CDPK (swCDPK) in all stages of embryo development (Anil et al., 2000). This gives credibility to swCDPK as a Ca²⁺ modulator, acting alone or in conjunction with CaM during sandalwood somatic embryogenesis. Although, W7 is a known CaM-antagonist, it also effectively inhibits CDPKs by blocking their C-terminal CaM-like domain (Harmon et al., 1986; DasGuptha, 1994). W7 inhibits the Ca²⁺-dependent autophosphorylation activity of the 55-kD swCDPK (Anil et al., 2000), and purified swCDPK exhibits the lowest 50% inhibition of initial activity for W7 inhibition among CDPKs characterized so far (V.S. Anil and K.S. Rad, unpublished data). Therefore it can be inferred that reduction in somatic embryogenesis in W7 culture treatments is due to blockage of downstream signaling pathway(s), wherein swCDPK and/or CaM are involved in perceiving the Ca²⁺ signal.

Although the enlarged clumps grown in exogenous Ca²⁺-chelated/deprived culture conditions exhibit functional protein synthesis machinery, they fail to accumulate detectable levels of swCDPK. This observation further suggests that exogenous Ca²⁺ is also necessary to induce optimal accumulation of swCDPK in the differentiating embryogenic cultures. This is not surprising since Ca²⁺ is known to induce and regulate the expression of CaM and CaM-like proteins that are players in Ca²⁺-mediated signal transduction. Furthermore the induction of CDPK expression by CaCl₂ and its inhibition by EGTA treatments have been reported earlier in mung bean and Sorghum plants (Botella et al., 1996; Pestenacz and Erdei, 1996). The culture treatment with ionophore also causes reduction in the accumulation of swCDPK in embryogenic cultures, suggesting that transitory cytosolic Ca²⁺ influxes may be of significance in the induction of swCDPK expression. The absence or undetectable levels of the enzyme probably contributed to the reduced embryogenic frequency under A23187 culture treatment.

This study thus presents evidence that culture conditions conducive for embryo development elicit a transient movement of exogenous Ca²⁺ into the cytosol of sandalwood pro-embryogenic cells. Culture treatments with Ca²⁺ antagonists further confirm the need for the exogenous Ca²⁺ pool not only for embryogenesis, but also for the expression of swCDPK. Further elucidation of the mechanism of stimulus-response coupling and identification of the different participants in the signaling pathway(s) would provide valuable insights into the process of plant embryogenesis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material

Embryogenic cultures were initiated from the endosperm of sandalwood (Santalum album) fruits in MS medium (Murashige and Skoog, 1962) supplemented with 1 mg L¹ each of benzylaminopurine and 2,4-D (callus initiation medium-MS₁). The endosperm-derived callus was subcultured onto MS medium containing 1 mg L¹ 2,4-D (callus proliferation medium-MS₂) for proliferation and for embryogenic induction. Cultures were maintained at 26°C ± 2°C under diffuse light conditions of 5 µEm² s¹. To isolate PEMs, the callus was suspended in liquid MS₂ medium and was strained through meshes of pore size 130, 80, and 50 µm, sequentially. Enriched PEMs were washed twice with the same medium. A packed cell volume (p.c.v.) of 250 µL of the washed cells was resuspended in liquid MS medium lacking 2,4-D and containing 2% (w/v) mannitol (embryo differentiation medium-MS₃) for embryo development. The culture was then agitated at 100 rpm on an orbital shaker under diffuse light conditions. Unless mentioned otherwise, 3% (w/v) Suc and 3.9 mM CaCl₂ were maintained in the medium.

Chemicals

Bepridil, A23187, W7, histone III-S, DMSO, dry DMSO, and chelex 100 were purchased from Sigma-Aldrich (St. Louis). Nifedipine and verapamil were procured from Boehringer Mannheim (Basel). Polyclonal antisoybean CDPK was a gift from A.C. Harmon (Department of Botany, University of Florida, Gainesville). Horseradish peroxidase-conjugated goat anti-rabbit IgG was purchased from Bangalore Genie (Bangalore, India). Radioactive isotopes [-³²P]ATP, [³⁵S]Met, and ⁴⁵CaCl₂ were purchased from BRIT (Hyderabad/Bombay, India).

⁴⁵Ca²⁺ Uptake by PEMs

Changes in the uptake of ⁴⁵Ca²⁺ with PEMs either retained in callus proliferation medium (MS₂) or subcultured into the differentiation medium (MS₃) were determined by first enriching PEMs in liquid MS₂ medium, followed by two washes in the same medium lacking Ca²⁺. Enriched PEMs, 250 µL of p.c.v. each, were subcultured into either liquid MS₂ or into liquid MS₃ medium, both lacking Ca²⁺. A control wherein PEMs were inoculated into MS₃ containing 1 mM EGTA was also included. Following the addition of 0.02 µCi of ⁴⁵Ca²⁺/mL of respective media, PEMs were incubated for 1 h at 26°C ± 2°C under diffuse light conditions. They were then washed twice with 1 mM Na EDTA (pH 8) for 10 min followed by similar washes with 2 mM LaCl₃ to remove cell wall-bound Ca²⁺. The incorporation of ⁴⁵Ca²⁺ was measured by liquid scintillation counting (LKB, Uppsala). Ten replicates were taken per treatment.

Washes with EDTA and lanthanum do not completely remove cell wall-bound ⁴⁵Ca²⁺. To determine the cell wall-sequestered ⁴⁵Ca²⁺, the washed PEMs were further treated with 2% (v/v) Triton X-100 overnight.

Measurement of Cytosolic Ca²⁺

PEMs were enriched in liquid MS₂ medium lacking minor nutrients. The loading of the Ca²⁺-sensitive fluorescent dye fura-2AM was carried out by incubating the PEMs in the above mentioned medium containing 0.02% (v/v) pluronic F-127 and 50 µM fura-2AM at 26°C ± 2°C on a gyratory shaker for 2 h in the dark. The 20% (v/v) stock of pluronic F-127 was prepared in dry DMSO. Following washing of PEMs to remove free fura-2AM, cells were viewed under a fluorescence microscope (Zeiss, Jena, Germany) to monitor loading of fura-2. The dye-loaded PEMs in MS₂ medium were made to settle on a coverslip coated with poly-Lys. Cytosolic-free Ca²⁺ was imaged by means of a laser scanning confocal microscope (TCS MP, single photon imaging system, Leica Microsystems, Wetzlar, Germany). Confocal time-based series of images were captured using excitation wavelengths of 351 and 361 nm and by recording emission at 512 nm. The fluorescence intensity in PEMs from the confocal images was determined after subtracting the background fluorescence in each experiment. Ratio (R) of emission from Ca²⁺-bound fura to emission from free fura (351 nm/361 nm excitation), and the ratios (R_max and R_min) from a predetermined calibration curve were used to estimate the [Ca²⁺]_cyt on line. The [Ca²⁺]_cyt in PEMs was determined when in MS₂ medium and also after perfusion of these PEMs with MS₃ or with MS₃ containing 1 mM EGTA.

Ca²⁺ calibration was achieved by incubating dye-loaded PEMs in 30 µM A23187 either with 2 mM Ca²⁺ or 5 mM EGTA in the external medium. Ratios of fluorescence in these PEMs represented the highest and lowest [Ca²⁺]_cyt, respectively, and were used in obtaining the calibration curve. The in vivo calibration (Gilroy et al., 1991) used in this study also reflects the responsiveness of the dye to changes in [Ca²⁺]_cyt.

Cell Culture Treatments

MS₃ containing a range of Ca²⁺ concentrations was prepared to determine the threshold concentration required for embryogenesis. Ca²⁺-Channel blockers, Ca²⁺ ionophore A23187, and CaM antagonist W7 were dissolved in 2% (v/v) DMSO. MS₃ was supplemented with increasing concentrations of one each of these pharmacological agents. Differentiation medium containing DMSO was included in the experiment as a control. Washed embryogenic cell clumps (250 µL of p.c.v.) were inoculated into 20 mL each of the above mentioned media. Each treatment given was in triplicate. Embryo development was monitored by microscopy. Only those PEMs that developed into bipolar-stage embryos were counted after a 21-d culture period.

Tissue Sectioning and Staining

Embryogenic cultures were fixed for 24 h in 70% (v/v) ethanol, acetic acid, and formaldehyde (90:5:5), upgraded in ethanol series, and infiltrated with paraffin wax in butanol at 50°C. Microtome sections of 12-µm thickness were cut and mounted on glass slides, deparaffinated with xylene, and stained with Heidenhain's hematoxylin. The sections were observed and photographed under a microscope (Zeiss).

Extraction of Soluble Proteins

The embryogenic tissue in each of the culture treatments was harvested after 21 d of culture incubation. Tissues were homogenized in liquid nitrogen using a pestle and mortar and suspended in the extraction buffer containing 2.5 mM EDTA, 20 mM Tris [tris(hydroxymethyl)aminomethane]-HCl, pH 7.2, and 1 mM phenylmethylsulfonyl fluoride. The extracts were spun at 13,650g for 30 min at 4°C in a refrigerated centrifuge (Sorvall Products, Newtown, CT). The supernatant containing the soluble proteins was used for further experimentation. Protein concentrations were determined according to the method of Bradford (1976) using bovine serum albumin as the standard. Protein extracts were mixed with Laemmli's sample buffer (Laemmli, 1970), boiled for 2 min, and resolved on a 10% (w/v) SDS polyacrylamide gel.

Protein Synthesis in Vivo

On d 21 of a typical differentiation cycle, 400 µL of p.c.v. of embryogenic cultures was withdrawn from MS₃ containing either optimal Ca²⁺ or 1 mM EGTA and incubated for 8 h in the presence of 50 µCi/mL of L-[³⁵S]Met. Labeled samples were centrifuged, and the pelleted embryogenic cultures were washed twice with liquid MS₃ to remove free label. The samples were pelleted again and resuspended in 2.5 mM EDTA, 20 mM Tris-HCl, pH 7.2, and 1 mM phenylmethylsulfonyl fluoride. The soluble proteins were extracted from these samples by sonication using a sonicator (VibroCell, Sonios and Materials Inc., Danbury, CT) equipped with a microtip in 20 5-s bursts at a setting of 5. After separating the cell debris, soluble proteins were resolved on an SDS polyacrylamide gel. The gel was treated with 2% (w/v) sodium salicylate in 30% (v/v) methanol for 30 min, dried, and an image of labeled proteins was obtained using the phosphor imager.

Protein Kinase Assay

Protein kinase activity was determined by measuring the incorporation of ³²P from [-³²P]ATP into histone III-S. In a total volume of 0.15 mL the assay mixture contained 1 mg mL¹ histone III-S, Ca/EGTA buffer (50 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], pH 7.2, 10 mM MgCl₂, and 0.25 mM EGTA) with or without 0.2 mM CaCl₂, and 10 µg of the protein sample. The reaction was initiated with addition of 10 nM [-³²P]ATP (5,000 nCi/pmol). The termination of the reaction, spotting of reaction mixture on glass microfiber filters (type C, Whatman, Maidstone, UK) and washing of the filters were carried out according to the method of Putnam-Evans et al. (1990). Counts were recorded on an LKB liquid scintillation counter.

Soluble proteins of embryogenic cultures subjected to various Ca²⁺ antagonist treatments were assayed for swCDPK autophosphorylation activity. The reaction was carried out in a total reaction volume of 30 µL, containing Ca/EGTA buffer with no exogenously added substrate and incubated for 20 min. The reaction was terminated by addition of Laemmli's sample buffer (Laemmli, 1970), boiled for 2 min, and resolved by SDS-PAGE overnight. The gel was Coomassie-stained to visualize the proteins, dried, and exposed to x-ray film (Kodak, Rochester, NY) for 24 h.

Immunostaining with Polyclonal Antisoybean CDPK

Protein extracts of embryogenic cultures subjected to various treatments were resolved by SDS-PAGE and transferred to nitrocellulose membrane (Towbin et al., 1979). The blot was blocked in rinse buffer (1× phosphate-buffered saline, pH 7.4, 0.05% [v/v] Tween 20) containing 1% (w/v) bovine serum albumin for 1 h. This was followed by incubation in dilution buffer (1× phosphate-buffered saline, pH 7.4, 0.5% [v/v] Tween 20) containing polyclonal antibodies directed against the CaM-like domain of soybean CDPK (Bachmann et al., 1996) at a concentration of 15 µg mL¹ for 3 h. Excess antibodies were removed by washing the blot with three changes of rinse buffer for 1 h. The blot was further incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase that was diluted to 1:1,000 in dilution buffer. After the removal of non-specifically bound secondary antibodies, swCDPK was visualized by incubation in citrate buffer containing diamino benzidene and hydrogen peroxide in dark.