Glycerol stimulation of chlorophyll synthesis, embryogenesis, and carboxylation and sucrose metabolism enzymes in nuceilar callus of. 'Hamlin' sweet orange.
Plant Cell, Tissue and Organ Culture 33: 75-80, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.
Glycerol stimulation of chlorophyll synthesis, embryogenesis, and carboxylation and sucrose metabolism enzymes in nuceilar callus of 'Hamlin' sweet orange Joseph C.V. Vu 1, Randall P. Niedz & George Yelenosky
United States Department of Agriculture, Agricultural Research Service, Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803, USA (lpresent address: USDA-ARS, Agronomy Physiology Laboratory, Building 164, University of Florida, Gainesville, FL 32611, USA) Received 4 December 1991; accepted in revised form 20 October 1992
Key words: carbon metabolism, Citrus tissue culture, glycerol Abstract Phosphoenolpyruvate carboxylase (PEPCase) and sucrose phosphate synthase (SPS) were active in nucellar calli of 'Hamlin' sweet orange, Citrus sinensis (L.) Osbeck, grown on media containing either 5% sucrose or 2% glycerol as the primary carbon source. Activities of the enzymes, however, were much higher in the glycerol-grown tissues than those grown in sucrose. Glycerol, in addition, stimulated embryogenesis and chlorophyll biosynthesis and initiated the onset of ribulose bisphosphate carboxylase-oxygenase (Rubisco) activity. These stimulatory effects, which were not observed in sucrose-grown calli, became more obvious when calli grown on glycerol-containing, agar-solidified medium were transferred and grown in liquid suspension medium containing 2% glycerol. Starch levels in both sucrose- and glycerol-grown tissues were as high as 30% of tissue dry weight. There were, however, higher concentrations of soluble sugars in sucrose-grown calli than those grown on glycerol. Stimulation of embryogenesis, chlorophyll synthesis, Rubisco onset, and activities of PEPCase and SPS by glycerol offered potential prospects in using this compound in citrus tissue cultures.
Abbreviations: PEPCase - phosphoenolpyruvate carboxylase, Rubisco - ribulose bisphosphate carboxylase-oxygenase, S P S - sucrose phosphate synthase
Introduction Sucrose is the most common carbon source in plant tissue and cell culture media (Ammirato et al. 1984; Gautheret 1955; Maretzki et al. 1974; Thompson & Thorpe 1987). In citrus, sucrose promotes greater callus growth than do other carbohydrates (Button 1978), but does not enhance somatic embryogenesis in callus cultures (Kochba et aI. 1982). In 1983, Ben-Hayyim &
Neumann reported the ability of several callus cultures of citrus to grow with glycerol as the sole carbon source, and a stimulatory effect of this polyalcohol on embryogenesis of these tissues. Identification of specific, potential targets in plant 'engineering' for more productive and/or stress-resistant citrus cultivars are integral parts of our long-term research goals. Plant tissue cultures offer attractive tools for a variety of
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76 physiological and biochemical studies, and carbon nutrition has been a major focal point ever since the early days of plant cell culture history (Gautheret 1955; Maretzki et al. 1974). The fundamental aspects of carbon utilization and metabolism in cell and callus cultures, as the external carbon source in the medium enters the metabolic pool of the tissues, have yet to be fully defined and understood for citrus as well as for other plant species. In this study, we compare some metabolic characteristics in nucellar callus and cell suspension cultures of 'Hamlin' sweet orange grown in medium containing sucrose or glycerol as the primary carbon source. The results indicate a stimulatory effect of glycerol on chlorophyll biosynthesis, embryogenesis, activities of phosphoenolpyruvate carboxylase (PEPCase) and sucrose phosphate synthase (SPS), and the onset of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The effects of glycerol and sucrose concentrations on growth of calli derived from several citrus cultivars have been previously investigated by other researchers (Ben-Hayyim & Neumann 1983).
Materials and methods
Plant material Embryogenic nucellar callus was induced from 6to 8-week old, post-pollination ovules of 'Hamlin' sweet orange (Citrus sinensis [L.] Osbeck), using the procedures of Kochba & Spiegel-Roy (1973). Following induction, the callus was subcultured every 28 days for 2 years at 27°C under cycles of 4-h light (15 Ixmol m -2 s -1 of cool-white fluorescent light) and 20-h darkness on an agarsolidified, 5% (w/v) sucrose Murashige-Tucker basal medium (MT), supplemented with 45 IxM benzylaminopurine (Kobayashi et al. 1983). For glycerol-grown calli, sucrose in the MT basal medium was replaced by 2% (v/v) glycerol. In this study, calli of the 35-day subcultures were removed at various intervals with a spatula free of culture medium and used directly for analyses without further treatment. Liquid suspension glycerol-grown cultures were initiated by transferring 1 g fresh weight of callus grown on MT + 2% glycerol for 28 days into 250-ml Erlenmeyer
flasks containing 50ml of MT basal liquid medium, with 2% glycerol substituted for 5% sucrose. The suspension cultures were incubated at 27°C on a gyratory shaker at 150 rpm and under very low-intensity, cool-white fluorescent light (1 to 2 i~molm-2s-~). Suspension tissues were collected after 14 days by vacuum filtration on # 4 Whatman paper disks and washed with deionized distilled water immediately before use.
PEPCase, Rubisco and SPS Extraction and assay of PEPCase and Rubisco were performed as described previously (Vu et al. 1985), using a ratio of 1:3 (fresh wt/volume) tissue-to-buffer. The incorporation of 14C into acid-stable products was determined by liquid scintillation spectrometry. Extraction and assay of SPS were accomplished according to a modified procedure of Huber et al. (1988). Approximately 1.2 g fresh weight tissue was extracted with 3 ml of an icechilled buffer containing 50raM Mops-NaOH (pH 7.5), 15 mM MgCI2, 1 mM EDTA, 2.5 mM DTT, and 0.1% (v/v) Triton X-100. The homogenate was centrifuged at 12,000g at 2°C for 1 min and the supernatant was rapidly desalted by centrifugal filtration on Sephadex G-25 column (Helmerhorst & Stokes 1980). Assay of SPS was performed at 25°C by injecting 90 ~1 at the enzyme extract to a 50 lxl of a reaction mixture containing 50 mM Mops-NaOH (pH 7.5), 2.5 mM DTI', 15 mM MgCl 2, 10 mM UDP-glucose, 10 mM fructose-6-P, and 40 mM glucose-6-P. The reaction was stopped after 10 rain with 140 Ixl of 1 N NaOH, and assay tubes were immersed in boiling water for 10 rain to destroy all unreacted fructose-6-P. After cooling, 0.5 rnl of 0.1% (w/v) resorcinol in 95% ethanol and 1.5 ml of 30% HC1 were added. The tubes were incubated at 80°C for 8 rain, cooled for 5 min in tap water, and absorbance was read at 520 nm. Blanks were run in parallel using the complete assay reaction mixture plus denatured enzyme.
Carbohydrates and chlorophyll Reducing sugars and sucrose were extracted from the tissues with hot 80% (v/v) ethanol, and concentrations were determined as previously
77 reported (Vu et al. 1990). The residual starch in the dry pellet was digested with amyloglucosidase, and glucose was determined in the supernatant. Chlorophyll was extracted with 80% (v/v) acetone and concentrations were determined according to Arnon (1949).
Results Activities of PEPCase and SPS in nucellar calli of 'Hamlin' sweet orange grown on agarsolidified basal medium containing 5% sucrose or 2% glycerol, as expressed on a basis of tissue dry weight, are shown in Fig. 1. PEPCase activities, when compared with day 0, were doubled at day 7 for both calli grown on 5% sucrose and 2% glycerol media, and tripled at days 21 and 35 for calli grown on 5% sucrose (Fig. 1A). Activities of PEPCase almost doubled every 7 days during the first 3 weeks of culture in calli grown on 2% glycerol-containing medium. 800-
A, PEPCase
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Activities of phosphoenolpyruvate carboxylase (PEP-
Case) (A) and sucrose phosphate synthase (SPS) (B) of 'Hamlin' sweet orange callus, grown for 35 days in 5% sucrose or 2% glycerol-containing medium. Data points are averages of 3 to 4 determinations; error bars denote SE. Points lacking error bars indicate that the SE was smaller than the symbol used.
PEPCase activities in calli grown on glycerolcontaining medium were about 8-fold at day 21, and 10-fold at day 35, when compared with day 0. PEPCase activity in sucrose-grown calli at day 35 was only about one-third of that in calli grown on glycerol-containing medium. No Rubisco activity was detected in either sucrose- or glycerolgrown calli during this growth period. Activity of SPS in 'Hamlin' calli grown on sucrose-containing medium was relatively unchanged during the first 14 days (Fig. 1B). Thereafter, activity increased about 70% at day 21, and decreased to about half of the initial (day 0) rate at day 35. In contrast, SPS activity in calli grown on glycerol-containing medium, as compared with day 0, increased about 60% at days 7 and 14, 280% at day 21, and 390% at day 35. Activity of SPS for glycerol-grown calli was higher, about 2-fold at day 21 and 10-fold at day 35, when compared with calli grown on sucrosecontaining medium. Patterns of PEPCase and SPS activities, when expressed on a basis of total soluble protein content, resembled those of the enzyme activities as shown in Fig. 1 with reference to tissue dry mass (data not presented). Total soluble protein content of calli grown on 5% sucrose did not change much during the 35-day subculture period, having average values of 3.7, 3.2, 2.5, 3.2, and 3.3 mgg -1 callus fresh weight for day 0, 7, 14, 21, and 35, respectively. Total soluble protein content of calli grown on 2% glycerol, however, averaged 3.7, 2.7, 2.9, 5.1, and 6.5mgg -~ callus fresh weight for day 0, 7, 14, 21, and 35, respectively. In addition, soluble proteins of 'Hamlin' suspension cultures, determined after 14 days of growth in 2% glycerol, were as high as 15.0 mgg -~ tissue fresh weight, (J.C.V. Vu, R.P. Niedz & G. Yelonosky unpubI.). Growth curves of our 'Hamlin' sweet orange calli grown in sucrose- and glycerol-containing media are presented in Fig. 2. Basically, these patterns of growth resembled those of 'Shamouti' sweet orange calli grown in 4% sucrose and 2% glycerol as reported by BenHayyim & Neumann (1983). In our study, 'Hamlin' calli grown on sucrose- and glycerol-containing medium for 35 days and transferred to fresh sucrose and glycerol medium restored rates of PEPCase and SPS, since activities of the enzymes determined at 21 and 28 days after the
78
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Fig. 2. Growth, as percent increase in fresh weight, of 'Hamlin' sweet orange nucellar callus on 5% sucrose or 2% glycerol-containing medium. Data points are averages of 3 (for glycerol) and 4 (for sucrose) culture plates; error bars denote SE. Points lacking error bars indicate that the SE was smaller than the symbol used.
subculture were comparable with those measured at similar periods of the immediate, previous subculture (see data presented in Fig. 1 for those corresponding days). Microscopic examina-
tion of calli two weeks after the initial transfer to agar-solidified glycerol-containing medium revealed the initiation of embryogenesis. Four weeks after the transfer to glycerol-containing medium, essentially all of the calli were converted into embryos; green color was visual, and chlorophyll and Rubisco were detectable in these glycerol-grown calli (Table 1). These phenomena, however, were not observed in sucrosegrown tissues. When the 4-week-old, glycerolgrown calli were successively transferred to fresh liquid medium containing 2% glycerol, the suspension cultures became much greener after 14 days; they contained more chlorophyll and had higher activity of Rubisco (Table 1). Also, activities of PEPCase and SPS in these suspension cultures were 25 and 40% higher, respectively, than the highest values as observed at 35 days (Fig. 1). In contrast, cultures grown on sucrose-containing medium remained pale white in color; chlorophyll and Rubisco were not detectable; PEPCase and SPS did not exceed beyond their maximum values as reported in Fig.
Table 1, Chlorophyll concentrations and Rubisco activities in nucellar cultures of 'Hamlin' sweet orange ~
Tissue
Rubisco Chlorophyll (Itg g-; dry wt)
(/x mol CO~g-~ dry wt h -~)
a
b
Total
A-28
8.4 (0.2)
5.9 (0.4)
14.3 (0.6)
3.4 (0.2)
S-14
147.3 (0.7)
71.3 (8.4)
219.6 (9.2)
5.8 (0.3)
Callus, which had been grown on an agar medium containing 5% sucrose for 28 days, was transferred to a fresh agar medium containing 2% glycerol for 28 days (A-28), and then to a suspension medium containing 2% glycerol for 14 days (S-14). Mean values and SE (shown in parentheses) of 2 (for chlorophyll) and 3 (for Rubisco) determinations are presented. Chlorophyll and Rubisco activities were not detected in cultures grown in 5% sucrose medium,
Table 2. Carbohydrate concentrations of 'Hamlin' sweet orange nucellar calli grown in medium containing 5% sucrose or 2%
glycerol. Days in fresh medium 0 7 14 21 35
Reducing sugars Sucrose (mg g-~ dry wt) 324.2(14.6) 274.5 (1.2) 252.1 (3.8) 180.6 (10.9) 139.7 (11.3)
Sucrose Glycerol
95.2(2.6) 72.9 (1.1) 74.8 (3.0) 66.2 (2.1)
Sucrose (mg g- ~dry wt) 80.1 (3.9) 119.2 (2.5) 108.7 (11.4) 125.3 (3.3) 43.2 (1.3)
Starch Glycerol
-" _a 14.5 (5.1) 3.2 (3.3)
Mean values and SE (shown in parentheses) of 3 to 5 determinations are presented. a Trace,
Sucrose (rag g -~ dry wt) 315.3 268.9 269.0 246.8 360.4
(2.7) (6.1) (4.2) (18.8) (3.8)
Glycerol
313.1(3.2) 314,1(6.9) 295.3 (6.9) 210.4 (5.0)
79 1, and microscopic observations did not reveal embryo formation or development in those tissues. Concentrations of soluble sugars and starch in 'Hamlin' cultures are shown in Table 2. Reducing sugars and sucrose were higher in 'Hamlin' calli grown in sucrose- than those grown on glycerol-containing medium. Sucrose concentrations in glycerol-grown tissues were only present either in trace or small amounts, when compared with the sucrose-grown tissues. Starch concentrations in both sucrose- and glycerol-grown tissues were more comparable. Starch concentration could account for up to 30% of tissue dry weights, even when glycerol was the only carbon source available for the cultures. Micrographs prepared from the cultures showed numerous starch granules in cells (not shown data).
Discussion
The capability of plant tissue cultures to use glycerol for growth was first reported in 1948 in carrot tissue (Gautheret 1955), and later in English sycamore (Scala & Semersky 1971), and a mutant cell line of tobacco (Chaleff & Parsons 1978). In 1983, stimulatory effects on somatic embryogenesis by glycerol were reported in nucellar callus cultures of several citrus cultivars (Ben-Hayyim & Neumann 1983). Although calli of various citrus can effectively utilize glycerol as well as sucrose for supporting growth, the calli grown on 2% glycerol, however, are always accompanied by embryogenesis, while there is no formation of embryos in the presence of sucrose (Ben-Hayyim & Neumann 1983). Somatic embryos are obtained upon transfer of callus derived from undeveloped ovules of sweet orange to the MT basal medium containing 5% lactose or 2% glycerol (Starrantino & Caponnetto 1991). In callus cultures of different citrus cultivars including 'Shamouti' and 'Navel' sweet oranges, sucrose supports embryogenesis to some extent only at low (0.2-1%) concentrations (Kochba et al. 1982). It has been reported that sucrose suppresses chlorophyll synthesis in carrot tissue cultures (Edelman & Hanson 1971). In our experiment, 'Hamlin' sweet orange cultures grown on medium containing 5% sucrose
were pale white, initiated no embryos, and no chlorophyll or Rubisco were detected. This was in contrast to the cultures grown in glycerolcontaining medium (Table 1) where embryogenesis was observed as early as 14 days after transfer. Phosphoenolpyruvate carboxylase has been reported to be involved during the developmental phases in many C 3 plants (Vu et al. 1985; and references therein). In flower buds and young developing fruits of sweet orange, PEPCase has been suggested as the enzyme involved in the synthesis of 4-carbon organic acids, among which malic acid is the most predominant form (Vu et al. 1985, 1990). In photosynthetically active carrot callus cultures, PEP carboxylation plays a significant role in their metabolism (Bender 1987). In 'Hamlin' calli grown in sucrose- and glycerol-containing media, both PEPCase and SPS were active, but with many-fold higher activities in the glycerol-cultured tissues than those grown in sucrose (Fig. 1). High activities of PEPCase and SPS in citrus calli indicate that the enzymes may play some important role on primary metabolism of these tissues. For calli grown on 2% glycerol medium, large increases in activities of PEPCase and SPS, in conjunction with increases in soluble proteins, as mostly observed at 21 and 35 days, would indicate that the increases in catalytic activities were at least partially due to de n o v o synthesis of the enzymes in these tissues. 'Hamlin' nucellar tissues also did have the capability to incorporate 14CO2 into acid-stable products, when NaH14CO3 was either added to the suspension liquid culture medium, or when calli grown on agar-solidified basal medium were exposed to 14CO2 derived from 14C-bicarbonate at room temperature (27°C) and under very lowintensity, cool-white fluorescent light (12 ~mol m -2 s -1 (J.C. Vu, unpubl.). In 'Hamlin' sweet orange, appreciable starch accumulation in the nucellar calli was sustained by either 5% sucrose or 2% glycerol as the sole carbon source (Table 2). With glycerol, 'Hamlin' calli were able to generate soluble sugars and starch from this polyalcohol. The modifying enzymatic pathways for this system should therefore be available, but it is not known whether these enzymes are substrate-inducible or always
80 present in the tissues. Preliminary analyses from our "Hamlin" sweet orange suspension cultures also indicate the presence of sucrose synthase (SS), with activity much higher for tissues grown in glycerol than sucrose medium. We hope that future research will reveal a possible role of PEPCase in these tissues, and to what extent SPS and SS may be involved in the metabolism of soluble sugars and starch, especially when glycerol is the sole carbon source for citrus tissue cultures. In addition, continued work is necessary to clarify the metabolic pathways leading to the formation of carbohydrates from glycerol, and the stimulatory effects of glycerol on somatic embryogenesis, chlorophyll synthesis, and Rubisco onset in citrus cultured tissues.
Acknowledgement We thank Delores Lomberk for skillful technical assistance in preparation and maintenance of the tissue culture lines.
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