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Comparison of photosynthetic characteristics of two photoautotrophic cell suspension cultures of soybean
Plant Science, 56 (1988) 69--74 Elsevier Scientific Publishers Ireland Ltd.
69
COMPARISON OF PHOTOSYNTHETIC CHARACTERISTICS OF TWO PHOTOAUTOTROPHIC CELL SUSPENSION CULTURES OF SOYBEAN
SUZANNE M.D. ROGERS and JACK M. WIDHOLM University of Illinois at Urbana-Champaign Turner Hall 1102 South Goodwin Avenue Urban~ IL 61801 (U.S.A./ (Received August 14th, 1987) (Revision received September 21st, 1987) (Accepted J a n u a r y 25th, 1988) A new photoautotrophic soybean suspension culture (SB1-P) was initiated within a 14-month period, and was characterized for the purpose of comparing the physiology with t h a t of a previously established soybean photoautotrophic strain, SB-P. SB1-P was found to have comparable Chl levels (1500--1700 ~g g-1 fresh weight), cell size (near 30 ~m in diameter) cell aggregate size (150-- 190 ~m diameter), chromosome n u m b e r (2n = 40), rates of growth (cell division time 4.5-- 5.0 days), net photosynthetic CO 2 fixation (19 and 36 ~emol mg -1 Chl h -1 at atmospheric levels of CO 2 and 21 and 2% 02, respectively) and oxygen evolution (74--76 pmol mg -1 Chl h-l), light-dependent and dark 14C02 fixation ( 4 9 - 5 4 and 3.5--3.7 pmol mg -~ Chl h -~, respectively) and oxygen inhibition of photosynthesis (47% inhibition at 21% 02 compared to photosynthesis at 2% 02 with atmospheric levels of C02). SB1-P was similar to SB-P with regard to the activation level of RuBPcase (40% activated in cells grown at 5% C02), low in vitro RuBPcase activity (25--30 pmol CO 2 fixed mg -~ Chl h-~), and a low ratio of RuBPcase(initial)/PEPcase (0.28--0.85). Traits shared by SB1-P and SB-P are a low photosynthetic capacity, an inability to grow at air levels of CO 2, high dark respiration, an elevated CO 2 compensation concentration in culture, and RuBPcase deactivation at air CO 2 levels. From these observations it was concluded t h a t many of the characteristics of photoautotrophic soybean cultures are caused by the conditions employed in culture, and are not due to the specific genotype of soybean cultured or to chance occurrence during initiation. The cultures which are actively growing exhibit several traits similar to those of developing leaf cells (active cell division, low photosynthetic capacity, elevated respiration, a low ratio of RuBPcase(initial)fPEPcase. This indicates the photoautotrophic cell physiology is more comparable to t h a t of developing leaf cells, rather than to that of mature leaf cells. K e y words: cell cultures; photoautotrophic; RuBP-carboxylase; soybean; photosynthesis
Introduction Photoautotrophic cells offer useful experimental systems for the study of plant physiology and recent investigations into the metabolism of photosynthetic cultures has conAbbreviations: Chl, chlorophyll; C a, photosynthesis by direct fixation of CO 2 by RuBPcase; C4, photosynthetic fixation of CO 2 by PEPcase; IR, infrared; SB1-P, photoautotrophic soybean line selected from Glycine max plant introduction PI-437833; SB1-M, SB1-P cells grown in medium with 1% sucrose; SB-P, photoautotrophic soybean line selected from Glycine max (L.) Merr variety Corsoy; 2,4-D, 2,4-dichlorophenoxyacetic acid; I-, the CO~ compensation concentration, which is the CO 2 concentration reached when the rates of CO z fixed by photosynthesis and CO 2 respired by cells are equal; RuBPcase, ribulose-l,5-bisphosphate carboxylase; PEPcase, phosphoenolpyruvate carboxylase
tributed to a better understanding of chloroplast-associated metabolic pathways and photosynthetic carbon assimilation [1-- 3]. Photoautotrophic cultures have unique characteristics different from those of intact mature leaves. Unlike mature leaves they require elevated levels of CO2 for growth, have active cell division, low photosynthetic rates, low RuBPcase activity, high respiration rates, and incorporate a large portion of CO2 via PEPcase. Although photoautotrophic cultures have been selected from a number of different plant species, no direct comparison of photosynthetic characteristics has been reported on independently derived photoautotrophic lines of the same species, let alone different genotypes. Different photosynthetic cell cultures exhibit variations in the level of photosynthetic potential,
0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
70 the amount of 14C labeling of C~ and C 4 photosynthetic products, Chl content, growth rates, and respiratory activity. It is not possible to discern whether some of the unique characteristics of these cultures are due to culture conditions, the physiological state of the cell, or the particular plant species cultured. To investigate this question a new photoautotrophic cell suspension line of soybean, Glycine max plant introduction 437833 was selected for comparison with an established soybean photoautotrophic line denoted SB-P (Glycine max (L.) Merr. var. Corsoy) [4]. In a previous paper we [5] described the photosynthetic and respiratory parameters of SB-P that has been cultured photoautotrophically for more than 4 years [4]. We now report on the characteristics of a new photoautotrophic soybean line, which was initiated within the last two years from a different soybean genotype. Materials and Methods
Culture initiation and development Stems of aseptically germinated soybean (G max PI-437833) seedlings were induced to form green callus on L2 medium [6] under 250 wE m -2 s -1 of continuous white light as described by Gray et al. [7]. Callus was provided by Y.Q. Guan in 6/85, when he was working in this laboratory. The medium contained 3% sucrose, 0.4 mg/l 2,4-D, 4.65 mg/1 naphthaleneacetic acid and 2.15 mg]l kinetin. Photomixotrophic callus was obtained by transferring the green callus to an agar solidified medium {0.8% agar) containing 1% sucrose (KNI%) [5] and growth under continuous white fluorescent light {250--300 wE m -2 s-l). Mixotrophic suspension cultures was produced by placing 1.5 g fresh wt. of mixotrophic callus in 50 ml of liquid K N I % medium in a 125-ml Erlenmeyer flask. KN medium is a modification of KT medium [4], which lacks Hepes buffer and all vitamins except thiamine [5]. The flasks were agitated at 130 rev./min on a gyratory shaker under continuous light. The cells were subcultured at 14-day intervals by inoculating 8 ml of cell suspension into 50 ml of medium. After the mixotrophic suspension, denoted
SB1-M, had been subcultured three times in KN1%, 2 g fresh wt. of cell were placed in 250ml flasks containing 80 ml of KN o medium (that lacks sucrose) in an atmosphere of 5% (v/v) CO2 as described by Horn et al. [4]. The new photoautotrophic soybean culture, designated SB1P, was subcultured at 2-week intervals, with an inoculum of 1.5 g fresh wt. of cells, for 5 months. At this time the inoculum was reduced to 0.5 g fresh wt. of cells. The photoautotrophic cultures were maintained as described previously
[5]. Growth measurements For measuring fresh weight, the cells were collected by vacuum filtration for 1 rain on 9.0 cm Whatman number 1 filter paper, in an 8.0 cm i.d. Buchner funnel. Chl was extracted from the cells with 80% (v/v) acetone and was measured spectrophotometrically [8]. Cell counting Duplicate flasks of cell suspensions were combined, and the cells collected by vacuum filtration. Three aliquots (0.22--0.26 g fresh wt.) of cells were suspended in 5 ml of 5% (w/v) chromium trioxide, shaken vigorously, and incubated 1 7 - 1 8 h at 26°C. To disperse the cells the suspension was again shaken vigorously, then drawn through a 23-gauge needle attached to a syringe six times. After dilution with a 7-fold volume of distilled water, the cells were counted using a Fuchs Rosenthal Ultra Plane counting chamber (0.2 mm depth). Each aliquot of cells was counted three times. Cell viability Cell viability was determined by mixing 1 drop of cell suspension with 1 drop of phenosafranine dye {0.1% in K N I % medium), as previously described [9]. Chromosome counting Five day old suspensions were incubated in 0.05% ~colchicine dissolved in KN ° medium for 2 h, then placed on Miracloth and washed with 50 ml of distilled water. Hydrolysis of cells was carried out in 0.2 N HC1 for 15 min at 25°C. The hydrolyzed cells were collected by centrifugation (100 × g, 5 min) and washed 5 times in
71
distilled water. The slides were prepared according to the method described by Kumar and Widholm [10] and the chromosomes of 50 cells were examined.
Photosynthesis, respiration and enzyme assays Photosynthetic oxygen evolution, lightdependent and dark 14CO2 fixation, net photosynthesis and dark respiration (as measured by IR gas exchange analysis), CO 2 compensation concentration (F), and enzyme assays were performed as described previously [5]. Results and Discussion
Culture establishment and growth Photomixotrophic cultures. To initiate a new photoautotrophic soybean suspension culture, friable green callus was initiated from stem tissue of the plant introduction (PI) 437833, which carries resistance to the soybean disease brown stem rot. A mixotrophic suspension culture, denoted SB1-M, was then obtained from the green callus. After 4 weeks of mixotrophic growth in K N I % , about 25% of the cell clumps in the SB1-M suspension culture gradually turned white. These clumps were determined to be dead by the phenosafranine viability stain. The 1% sucrose in the K N I % medium has been shown to be growth limiting compared to 3% sucrose in the medium [4]. Six weeks after initiation, the Chl content of the SB1-M cells had increased from 52 to 102 ~g Chl g-1 fresh wt. and the culture was composed predominately of viable green cell aggregates. During the first 33 weeks of mixotrophic growth (15 transfers) the Chl content of SB1-M cells increased from 52 to 700 ~g Chl g-1 fresh wt, and has remained stable at about 650--775 ~g Chl g-~ fresh wt. (Fig. 1). Photoautotrophic cultures. To obtain the photoautotrophic culture, SP1-P, the SB1-M cells were transferred to KN o medium which has no sucrose under at atmosphere of 5% CO 2 and constant light. The majority {90%} of the cells subcultured to photoautotrophic conditions turned white and died while cell aggregates able to photosynthesize and grow remained dark green. As the green cells divid-
1800
_
418ffi
.! ~/eeks o f Growth
Fig. 1. Chl levels (per g fresh wt.) of SBI-M (D) and SBI-P (m) cultures during the first 50 weeks of photomixotrophic and photoautotrophic growth, respectively. The medium for mixotrophic growth contained 1% sucrose while that for photoautotrophic growth had none.
ed and grew they made up an increasing proportion of the culture so after 6 weeks of photoautotrophic growth the culture was composed of about 20% viable cell clumps. By 12 weeks of growth this figure had increased to about 60% viable aggregates. After 35 weeks of growth the increase in Chl content (Fig. 1) reflected the increased proportion of the culture which was composed of viable green cells (90%). Five months after initiation, the SB1-P cells exhibited a mean doubling time of 4.5-- 5.0 days and the Chl content was 458--622 ~g Chl g-1 fresh wt. The pH of the culture medium quickly dropped from 5.2 to 4.5 following subculture and remained stable until the end of the growth period, when it increased to a final pH of about 4.7. The Chl content of the SB1-P cells stabilized, after 8 months in culture, at 1500--1700 ~g Chl g-1 fresh weight (Fig. 1) and has remained at this level. The SB1-P Chl content is comparable to that in SB-P (1500--1900 ~g Chl g-1 fresh wt) [5]. At this time the SB1-P culture was comprised of small cell aggregates, about 30--50 cells/aggregate, with a cell viability of greater than 99%. When the chromosome numbers of both lines were measured at this time, the SB1-
72 P cells were largely normal (2n = 40), as were the SB-P cells (2n = 40). Only 2% of the SB1-P and SB-P cells showed variation from this normal soybean chromosome number. In a previous study, Chowdhury and Widholm [11] found the same chromosome number for SB-P. This is unusual since cell cultures often show aberrant chromosome numbers. Characterization. Currently the SB1-P cells have been grown photoautotrophically for 2 years and possess physiological properties quite comparable to SB-P which have been grown photoautotrophically for about 5 years [4]. SB1-P was shown to have an absolute requirement for an atmosphere of elevated CO 2 {data not shown), regreens rapidly in the light after being bleached in the dark, has thiamine as the sole vitamin requirement, and grows linearly for at least 18 days. The 18-day-old cultures grew to an average of 3.6 g fresh wt. flask -1, compared to 4.7 g fresh wt. flask -1 for 18-day-old SB-P cultures [5] with an initial inoculum of 0.3 g fresh wt. The lower fresh weight accumulation means that SB1-P has a slightly longer doubling time than SB-P (4.5--5.0 vs. 3.6 --4.0 days, respectively). During the first 2 days SB1-P cell number (per g fresh wt.) decreases from 90 × 106 cells g-1 fresh wt. to near 50 × 106, indicating an increase in cell size, while that of SB-P remains unchanged (60 × 106 cells g-~ fresh wt.). Thereafter both lines show a linear increase in cells per gram fresh weight, with SB1-P and SB-P cells reaching a maximum of 125 and 96 × 106 cell g-1 fresh wt., respectively between day 5 and 8. SB1-P cells are comparable both in cell size, and pattern of cell number (per gram fresh weight) to SB-P cells. The early part of the growth period of SB1-P (day 2--6} is characterized by decreased Chl content and increased soluble protein levels, as is seen in SB-P [5]. The SB1-P cell Chl content decreases by nearly 50O/o and the protein content increases by nearly 25% on fresh weight basis during this period. This part of the growth period corresponds to the rapid synthesis phase.
Photosynthetic activity as measured by oxygen evolution ( 7 4 - 76 t~mol mg -1 Chl h -1 with 1.5 mM HCO3-), and the incorporation of 14C02 ( 4 9 - 5 4 t~mol mg -1 Chl h -1 with 5 mM HC08-) document that the photosynthetic activities of PI-P cells are similar to those of SB-P. SB-P cells had photosynthetic oxygen evolution and 14C02 fixation rates of 5 3 - 1 0 0 and 3 3 - 6 6 t~mol mg -~ Chl h -1, under the same conditions respectively [5]. The ranges are due to the decrease in Chl content, which regularly occurs early in the subculture period. Dark ~4C02 fixation was low, being not more than 7.5% of photosynthetic 14C02 fixation in SB1-P, compared to about 8% in SB-P [5]. Photosynthesis of SB1-P cells measured by IR gas analysis as net CO 2 fixation was inhibited by more than 40% at 21% oxygen compared to photosynthesis in 2% oxygen at near atmospheric levels of CO 2. The values for 4 and 10 day cells was 36 and 29 t~mol CO 2 mg -~ Chl h -1 in 2% 02, respectively. In a similar experiment SB-P photosynthesis was inhibited about 50% [5]. Thus photosynthesis of SB1-P cells, like that of SB-P cells, was found to be comparably sensitive to oxygen. SB1-P cells have stable photosynthetic rates, which are comparable to the rates of SB-P cells [5]. However an assay on 5 day SB1-P cells (data not shown) showed they have lower photosynthetic rates and higher dark respiration rates at this time. This is comparable to the case with SB-P, which has stable photosynthetic (10 and 22 t~mol CO 2 fixed mg -~ Chl h -~ at 21% and 2% 02 , respectively) and dark respiration rates (14 tamol CO 2 evolved mg -1 Chl h -a at 21% and 20/0 02). However again there is a dip in photosynthetic rates (evolved 3 tamol CO 2 mg -1 Chl h -~ at 21% 02 in the light, fixed 4 t~mol CO 2 mg -1 Chl h -1 at 2% 02 in the light) and a large peak of dark respiration (41 tzmol CO 2 evolved mg -~ Chl h -~ at 21% or 2% 02) in 4-day-old SB-P cells. This large decrease in photosynthesis and peak of dark respiration, which occur on different days in the two cell lines, is presumably due to the difference in the doubling times of SB1-P and SB-P cells (about 5 and 4 days, respectively}.
73 The ability of SB1-P cells to carry out net photosynthesis in air indicated a COs compensation concentration (I-) below air levels o f CO2 (361- 373 ppm). The I- of SB1-P cells was found to be 231 _+ 8 ppm under liquid culture conditions, a [- almost identical to that of SB-P cells under the same conditions (230 ± 25 ppm) [5]. The I- of SB1-P cells removed from the liquid medium spread as a thin layer in a Petri dish and analyzed, was 91 _+ 6 ppm COs. This value was also virtually the same as the [- for SB-P cells removed from the medium (90 _+ 15 ppm) indicating that presence of the liquid medium has a comparable effect on the photosynthetic efficiency of both lines. Neither line will grow in air, however, so net photosynthesis over the entire culture period is inadequate to support cell growth and maintenance under such conditions. Measurements of RuBPcase activity document that there are only small changes in activity during the growth period. RuBPcase initial (in vivo, about 25--35 ~mol CO 2 mg -1 Chl h -1) and total (that which is present after preincubation with COs and Mg 2÷to fully activate the enzyme, about 50--80 pmol COs mg -1 Chl h -i) were quite comparable to those in SB-P cells through the culture cycle where respective initial and total rates of about 40 and 80 pmol mg -~ CO2 Chl h -1 were found. In comparison to the activities of soybean leaf RuBPcase (initial and total rates of 392 and 516 ~mol CO s mg -~ Chl h -1) [5], it is evident that the low RuBPcase activities, of both soybean photoautotrophs, are apparently a major cause of the low photosynthetic capacity of the cells. PEPcase activity in SB1-P cells showed the characteristic peak in activity early in the growth period (day 4) and then declined to a uniform level. The peak activity was about 110 pmol COs m g -1 Chl h -1 while the usual level was about 40. Due to the changes in PEPcase, the ratio of RuBPcase(I)/PEPcase activity was lowest early in the growth period, about 0.3, and then increased to a maximum of about 0.85 during the latter part of growth. These ratios were slightly lower, but comparable to, the values
measured in SB-P (RuBPcase(I)/PEPcase of 0.5 -1.5). PEPcase activity, whether expressed on a Chl basis or on a soluble protein basis, was similar to the activities found in SB-P (30--90 ~mol CO2 mg -1 Chl hbl). Low CO2 had the same effect on RuBPcase activation in SB1-P cells as it did in SB-P cells. RuBPcase initial activity, as a percentage of total activity was about 40% in SB1-P cells grown in an atmosphere of 5% CO e, which was comparable to the 50% activation level in SB-P cells in 5% COe. In SB1-P cells exposed to air for 24 h, the RuBPcase was only about 20% activated in comparison to 15% activation in SB-P cells exposed to air. These findings are in agreement with the observation from SB-P cultures where the deactivation of RuBPcase at low COs (only about 14% activated) results in severely decreased photosynthetic capacity [5], which would apparently limit photosynthesis and in turn would limit the cells ability to grow in air. Conclusions Until now no one has compared the photosynthetic characteristics of different photoautotrophic cell lines derived from the same plant species. For this purpose a second photoautotrophic soybean line, SB1-P, was established, characterized and compared to the original photoautotrophic soybean line SB-P. This study allowed us to determine if the unusual photosynthetic characteristics of SB-P in comparison to mature leaves, would be present in other soybean photoautotrophic lines, or if such characteristics were unique to SB-P. Analysis showed no significant differences in the characteristics of SB1-P compared to SB-P. Grown photoautotrophically in an atmosphere of 5% C02, SB1-P has comparable Chl levels, cell size, cell aggregate size, chromosome number, rates of growth, net photosynthetic CO2 fixation and oxygen evolution, light-dependent and dark 14C02 fixation, dark respiration and oxygen inhibition of photosynthesis. SB1-P is comparable to SB-P with regard to percent
74
RuBPcase activation, low RuBPcase activity, and PEPcase activity. Both lines are unable to grow at air levels of CO2, have high respiration, an elevated C02 compensation concentration, and show deactivation of RuBPcase at ambient levels of CO2. We conclude that SB-P and SB1-P are very similar physiologically. From the results of this investigation it is concluded that many of the unusual characteristics of these lines are the result of the conditions employed in culture, and are not due to the use of any specific soybean genotype. Studies with soybean plants have shown that decreasing the light intensity to 20% full sunlight decreased the activation state of RuBPcase from 94% to 74% [12] and increasing the CO2 level to 800 ~lfl also decreased the RuBPcase activity [13]. The photoautotrophic cultures are also grown under continuous light conditions to attempt to maximize photosynthesis unlike plants which normally undergo light and dark periods. However, when the SB-P cells were grown with a 10-h dark period, the cell growth slowed and they died within a few weeks (M. Horn, B. Martin and J.M. Widholm, unpublished). The physiology of rapidly growing photoautotrophic cells would be expected to be comparable to that of developing leaf cells. This is indicated by the active cell division, high dark respiration, low RuBPcase activity, elevated [-, and low photosynthetic efficiency, which are characteristics common between SB1-P and SB-P.
References 1
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Acknowledgements Supported in part by funds from the Illinois Agricultural Experimental Station, Agrigenetics and by the McKnight Interdisciplinary Photosynthesis Research Program.
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W. Husemann, H. Herzbeck and H. Robenek, Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubram from different phases of batch growth. Physiol. Plant., 62 {1984) 349 -- 355. F. Sato, K. Nishida and Y. Yamada, Activities of carboxylation enzymes and products of 14C02 fixation in photoautotrophically cultured cells. Plant Sci. Lett., 20 (1980) 91-- 97. S. Seeni and A. Gnanam, Growth of photoautotrophic cells of peanut (Arachis hypogaea L.) in still nutrient medium. Plant Physiol., 70 (1982) 815--822. M.E. Horn, J.H. Sherrard and J.M. Widholm, Photoautotrophic growth of soybean cells in suspension culture. I. Establishment of photoautotrophic cultures. Plant Physiol., 72 (1983) 426--429. S.M.D. Rogers, W.L. Ogren and J.M. Widholm, Photosynthetic characteristics of a photoautotrophic cell suspension culture of soybean. Plant Physiol., (1987) 1451 -- 1456. G.C. Phillips and G.B. Collins, Induction and development of somatic embryos from cell suspension cultures of soybean. Plant Cell Tiss. Org. Culture, 1 {1981) 1 2 3 129. L.E. Gray, Y.Q. Guan and J.M. Widholm, Reaction of soybean callus to culture filtrates of Phialophora gregata, Plant Sci., 47 (1986) 45-- 55. D.I. Arnon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24 (1949) 1-- 10. J. Widholm, The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol., 47 (1972} 189-194. P. Kumar and J.M. Widholm, Techniques for chromosome analysis of carrot culture cells. Plant Mol. Biol. Rep., 2 (1984) 37-- 42. V.K. Chowdhury and J.M. Widholm, Callus production from photoautotrophic soybean cell culture protoplasts. Plant Cell Rep., 4 (1985} 2 8 9 - 292. R.S. Torisky and J.C. Servaites, Effect of irradiance during growth of Glycine max on photosynthetic capacity and percent activation of ribulose, 1,5-bisphosphate carboxylase. Photosynth. Res., 5 {1984) 251 - 261. C.V. Vu, L.H. Allen and G. Bowes, Effects of light and elevated atmospheric CO 2 on the ribulose bisphosphate carboxylase activity and ribulose bisphosphate level in soybean leaves. Plant Physiol., 73 {1983} 7 2 9 734.
69
COMPARISON OF PHOTOSYNTHETIC CHARACTERISTICS OF TWO PHOTOAUTOTROPHIC CELL SUSPENSION CULTURES OF SOYBEAN
SUZANNE M.D. ROGERS and JACK M. WIDHOLM University of Illinois at Urbana-Champaign Turner Hall 1102 South Goodwin Avenue Urban~ IL 61801 (U.S.A./ (Received August 14th, 1987) (Revision received September 21st, 1987) (Accepted J a n u a r y 25th, 1988) A new photoautotrophic soybean suspension culture (SB1-P) was initiated within a 14-month period, and was characterized for the purpose of comparing the physiology with t h a t of a previously established soybean photoautotrophic strain, SB-P. SB1-P was found to have comparable Chl levels (1500--1700 ~g g-1 fresh weight), cell size (near 30 ~m in diameter) cell aggregate size (150-- 190 ~m diameter), chromosome n u m b e r (2n = 40), rates of growth (cell division time 4.5-- 5.0 days), net photosynthetic CO 2 fixation (19 and 36 ~emol mg -1 Chl h -1 at atmospheric levels of CO 2 and 21 and 2% 02, respectively) and oxygen evolution (74--76 pmol mg -1 Chl h-l), light-dependent and dark 14C02 fixation ( 4 9 - 5 4 and 3.5--3.7 pmol mg -~ Chl h -~, respectively) and oxygen inhibition of photosynthesis (47% inhibition at 21% 02 compared to photosynthesis at 2% 02 with atmospheric levels of C02). SB1-P was similar to SB-P with regard to the activation level of RuBPcase (40% activated in cells grown at 5% C02), low in vitro RuBPcase activity (25--30 pmol CO 2 fixed mg -~ Chl h-~), and a low ratio of RuBPcase(initial)/PEPcase (0.28--0.85). Traits shared by SB1-P and SB-P are a low photosynthetic capacity, an inability to grow at air levels of CO 2, high dark respiration, an elevated CO 2 compensation concentration in culture, and RuBPcase deactivation at air CO 2 levels. From these observations it was concluded t h a t many of the characteristics of photoautotrophic soybean cultures are caused by the conditions employed in culture, and are not due to the specific genotype of soybean cultured or to chance occurrence during initiation. The cultures which are actively growing exhibit several traits similar to those of developing leaf cells (active cell division, low photosynthetic capacity, elevated respiration, a low ratio of RuBPcase(initial)fPEPcase. This indicates the photoautotrophic cell physiology is more comparable to t h a t of developing leaf cells, rather than to that of mature leaf cells. K e y words: cell cultures; photoautotrophic; RuBP-carboxylase; soybean; photosynthesis
Introduction Photoautotrophic cells offer useful experimental systems for the study of plant physiology and recent investigations into the metabolism of photosynthetic cultures has conAbbreviations: Chl, chlorophyll; C a, photosynthesis by direct fixation of CO 2 by RuBPcase; C4, photosynthetic fixation of CO 2 by PEPcase; IR, infrared; SB1-P, photoautotrophic soybean line selected from Glycine max plant introduction PI-437833; SB1-M, SB1-P cells grown in medium with 1% sucrose; SB-P, photoautotrophic soybean line selected from Glycine max (L.) Merr variety Corsoy; 2,4-D, 2,4-dichlorophenoxyacetic acid; I-, the CO~ compensation concentration, which is the CO 2 concentration reached when the rates of CO z fixed by photosynthesis and CO 2 respired by cells are equal; RuBPcase, ribulose-l,5-bisphosphate carboxylase; PEPcase, phosphoenolpyruvate carboxylase
tributed to a better understanding of chloroplast-associated metabolic pathways and photosynthetic carbon assimilation [1-- 3]. Photoautotrophic cultures have unique characteristics different from those of intact mature leaves. Unlike mature leaves they require elevated levels of CO2 for growth, have active cell division, low photosynthetic rates, low RuBPcase activity, high respiration rates, and incorporate a large portion of CO2 via PEPcase. Although photoautotrophic cultures have been selected from a number of different plant species, no direct comparison of photosynthetic characteristics has been reported on independently derived photoautotrophic lines of the same species, let alone different genotypes. Different photosynthetic cell cultures exhibit variations in the level of photosynthetic potential,
0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
70 the amount of 14C labeling of C~ and C 4 photosynthetic products, Chl content, growth rates, and respiratory activity. It is not possible to discern whether some of the unique characteristics of these cultures are due to culture conditions, the physiological state of the cell, or the particular plant species cultured. To investigate this question a new photoautotrophic cell suspension line of soybean, Glycine max plant introduction 437833 was selected for comparison with an established soybean photoautotrophic line denoted SB-P (Glycine max (L.) Merr. var. Corsoy) [4]. In a previous paper we [5] described the photosynthetic and respiratory parameters of SB-P that has been cultured photoautotrophically for more than 4 years [4]. We now report on the characteristics of a new photoautotrophic soybean line, which was initiated within the last two years from a different soybean genotype. Materials and Methods
Culture initiation and development Stems of aseptically germinated soybean (G max PI-437833) seedlings were induced to form green callus on L2 medium [6] under 250 wE m -2 s -1 of continuous white light as described by Gray et al. [7]. Callus was provided by Y.Q. Guan in 6/85, when he was working in this laboratory. The medium contained 3% sucrose, 0.4 mg/l 2,4-D, 4.65 mg/1 naphthaleneacetic acid and 2.15 mg]l kinetin. Photomixotrophic callus was obtained by transferring the green callus to an agar solidified medium {0.8% agar) containing 1% sucrose (KNI%) [5] and growth under continuous white fluorescent light {250--300 wE m -2 s-l). Mixotrophic suspension cultures was produced by placing 1.5 g fresh wt. of mixotrophic callus in 50 ml of liquid K N I % medium in a 125-ml Erlenmeyer flask. KN medium is a modification of KT medium [4], which lacks Hepes buffer and all vitamins except thiamine [5]. The flasks were agitated at 130 rev./min on a gyratory shaker under continuous light. The cells were subcultured at 14-day intervals by inoculating 8 ml of cell suspension into 50 ml of medium. After the mixotrophic suspension, denoted
SB1-M, had been subcultured three times in KN1%, 2 g fresh wt. of cell were placed in 250ml flasks containing 80 ml of KN o medium (that lacks sucrose) in an atmosphere of 5% (v/v) CO2 as described by Horn et al. [4]. The new photoautotrophic soybean culture, designated SB1P, was subcultured at 2-week intervals, with an inoculum of 1.5 g fresh wt. of cells, for 5 months. At this time the inoculum was reduced to 0.5 g fresh wt. of cells. The photoautotrophic cultures were maintained as described previously
[5]. Growth measurements For measuring fresh weight, the cells were collected by vacuum filtration for 1 rain on 9.0 cm Whatman number 1 filter paper, in an 8.0 cm i.d. Buchner funnel. Chl was extracted from the cells with 80% (v/v) acetone and was measured spectrophotometrically [8]. Cell counting Duplicate flasks of cell suspensions were combined, and the cells collected by vacuum filtration. Three aliquots (0.22--0.26 g fresh wt.) of cells were suspended in 5 ml of 5% (w/v) chromium trioxide, shaken vigorously, and incubated 1 7 - 1 8 h at 26°C. To disperse the cells the suspension was again shaken vigorously, then drawn through a 23-gauge needle attached to a syringe six times. After dilution with a 7-fold volume of distilled water, the cells were counted using a Fuchs Rosenthal Ultra Plane counting chamber (0.2 mm depth). Each aliquot of cells was counted three times. Cell viability Cell viability was determined by mixing 1 drop of cell suspension with 1 drop of phenosafranine dye {0.1% in K N I % medium), as previously described [9]. Chromosome counting Five day old suspensions were incubated in 0.05% ~colchicine dissolved in KN ° medium for 2 h, then placed on Miracloth and washed with 50 ml of distilled water. Hydrolysis of cells was carried out in 0.2 N HC1 for 15 min at 25°C. The hydrolyzed cells were collected by centrifugation (100 × g, 5 min) and washed 5 times in
71
distilled water. The slides were prepared according to the method described by Kumar and Widholm [10] and the chromosomes of 50 cells were examined.
Photosynthesis, respiration and enzyme assays Photosynthetic oxygen evolution, lightdependent and dark 14CO2 fixation, net photosynthesis and dark respiration (as measured by IR gas exchange analysis), CO 2 compensation concentration (F), and enzyme assays were performed as described previously [5]. Results and Discussion
Culture establishment and growth Photomixotrophic cultures. To initiate a new photoautotrophic soybean suspension culture, friable green callus was initiated from stem tissue of the plant introduction (PI) 437833, which carries resistance to the soybean disease brown stem rot. A mixotrophic suspension culture, denoted SB1-M, was then obtained from the green callus. After 4 weeks of mixotrophic growth in K N I % , about 25% of the cell clumps in the SB1-M suspension culture gradually turned white. These clumps were determined to be dead by the phenosafranine viability stain. The 1% sucrose in the K N I % medium has been shown to be growth limiting compared to 3% sucrose in the medium [4]. Six weeks after initiation, the Chl content of the SB1-M cells had increased from 52 to 102 ~g Chl g-1 fresh wt. and the culture was composed predominately of viable green cell aggregates. During the first 33 weeks of mixotrophic growth (15 transfers) the Chl content of SB1-M cells increased from 52 to 700 ~g Chl g-1 fresh wt, and has remained stable at about 650--775 ~g Chl g-~ fresh wt. (Fig. 1). Photoautotrophic cultures. To obtain the photoautotrophic culture, SP1-P, the SB1-M cells were transferred to KN o medium which has no sucrose under at atmosphere of 5% CO 2 and constant light. The majority {90%} of the cells subcultured to photoautotrophic conditions turned white and died while cell aggregates able to photosynthesize and grow remained dark green. As the green cells divid-
1800
_
418ffi
.! ~/eeks o f Growth
Fig. 1. Chl levels (per g fresh wt.) of SBI-M (D) and SBI-P (m) cultures during the first 50 weeks of photomixotrophic and photoautotrophic growth, respectively. The medium for mixotrophic growth contained 1% sucrose while that for photoautotrophic growth had none.
ed and grew they made up an increasing proportion of the culture so after 6 weeks of photoautotrophic growth the culture was composed of about 20% viable cell clumps. By 12 weeks of growth this figure had increased to about 60% viable aggregates. After 35 weeks of growth the increase in Chl content (Fig. 1) reflected the increased proportion of the culture which was composed of viable green cells (90%). Five months after initiation, the SB1-P cells exhibited a mean doubling time of 4.5-- 5.0 days and the Chl content was 458--622 ~g Chl g-1 fresh wt. The pH of the culture medium quickly dropped from 5.2 to 4.5 following subculture and remained stable until the end of the growth period, when it increased to a final pH of about 4.7. The Chl content of the SB1-P cells stabilized, after 8 months in culture, at 1500--1700 ~g Chl g-1 fresh weight (Fig. 1) and has remained at this level. The SB1-P Chl content is comparable to that in SB-P (1500--1900 ~g Chl g-1 fresh wt) [5]. At this time the SB1-P culture was comprised of small cell aggregates, about 30--50 cells/aggregate, with a cell viability of greater than 99%. When the chromosome numbers of both lines were measured at this time, the SB1-
72 P cells were largely normal (2n = 40), as were the SB-P cells (2n = 40). Only 2% of the SB1-P and SB-P cells showed variation from this normal soybean chromosome number. In a previous study, Chowdhury and Widholm [11] found the same chromosome number for SB-P. This is unusual since cell cultures often show aberrant chromosome numbers. Characterization. Currently the SB1-P cells have been grown photoautotrophically for 2 years and possess physiological properties quite comparable to SB-P which have been grown photoautotrophically for about 5 years [4]. SB1-P was shown to have an absolute requirement for an atmosphere of elevated CO 2 {data not shown), regreens rapidly in the light after being bleached in the dark, has thiamine as the sole vitamin requirement, and grows linearly for at least 18 days. The 18-day-old cultures grew to an average of 3.6 g fresh wt. flask -1, compared to 4.7 g fresh wt. flask -1 for 18-day-old SB-P cultures [5] with an initial inoculum of 0.3 g fresh wt. The lower fresh weight accumulation means that SB1-P has a slightly longer doubling time than SB-P (4.5--5.0 vs. 3.6 --4.0 days, respectively). During the first 2 days SB1-P cell number (per g fresh wt.) decreases from 90 × 106 cells g-1 fresh wt. to near 50 × 106, indicating an increase in cell size, while that of SB-P remains unchanged (60 × 106 cells g-~ fresh wt.). Thereafter both lines show a linear increase in cells per gram fresh weight, with SB1-P and SB-P cells reaching a maximum of 125 and 96 × 106 cell g-1 fresh wt., respectively between day 5 and 8. SB1-P cells are comparable both in cell size, and pattern of cell number (per gram fresh weight) to SB-P cells. The early part of the growth period of SB1-P (day 2--6} is characterized by decreased Chl content and increased soluble protein levels, as is seen in SB-P [5]. The SB1-P cell Chl content decreases by nearly 50O/o and the protein content increases by nearly 25% on fresh weight basis during this period. This part of the growth period corresponds to the rapid synthesis phase.
Photosynthetic activity as measured by oxygen evolution ( 7 4 - 76 t~mol mg -1 Chl h -1 with 1.5 mM HCO3-), and the incorporation of 14C02 ( 4 9 - 5 4 t~mol mg -1 Chl h -1 with 5 mM HC08-) document that the photosynthetic activities of PI-P cells are similar to those of SB-P. SB-P cells had photosynthetic oxygen evolution and 14C02 fixation rates of 5 3 - 1 0 0 and 3 3 - 6 6 t~mol mg -~ Chl h -1, under the same conditions respectively [5]. The ranges are due to the decrease in Chl content, which regularly occurs early in the subculture period. Dark ~4C02 fixation was low, being not more than 7.5% of photosynthetic 14C02 fixation in SB1-P, compared to about 8% in SB-P [5]. Photosynthesis of SB1-P cells measured by IR gas analysis as net CO 2 fixation was inhibited by more than 40% at 21% oxygen compared to photosynthesis in 2% oxygen at near atmospheric levels of CO 2. The values for 4 and 10 day cells was 36 and 29 t~mol CO 2 mg -~ Chl h -1 in 2% 02, respectively. In a similar experiment SB-P photosynthesis was inhibited about 50% [5]. Thus photosynthesis of SB1-P cells, like that of SB-P cells, was found to be comparably sensitive to oxygen. SB1-P cells have stable photosynthetic rates, which are comparable to the rates of SB-P cells [5]. However an assay on 5 day SB1-P cells (data not shown) showed they have lower photosynthetic rates and higher dark respiration rates at this time. This is comparable to the case with SB-P, which has stable photosynthetic (10 and 22 t~mol CO 2 fixed mg -~ Chl h -~ at 21% and 2% 02 , respectively) and dark respiration rates (14 tamol CO 2 evolved mg -1 Chl h -a at 21% and 20/0 02). However again there is a dip in photosynthetic rates (evolved 3 tamol CO 2 mg -1 Chl h -~ at 21% 02 in the light, fixed 4 t~mol CO 2 mg -1 Chl h -1 at 2% 02 in the light) and a large peak of dark respiration (41 tzmol CO 2 evolved mg -~ Chl h -~ at 21% or 2% 02) in 4-day-old SB-P cells. This large decrease in photosynthesis and peak of dark respiration, which occur on different days in the two cell lines, is presumably due to the difference in the doubling times of SB1-P and SB-P cells (about 5 and 4 days, respectively}.
73 The ability of SB1-P cells to carry out net photosynthesis in air indicated a COs compensation concentration (I-) below air levels o f CO2 (361- 373 ppm). The I- of SB1-P cells was found to be 231 _+ 8 ppm under liquid culture conditions, a [- almost identical to that of SB-P cells under the same conditions (230 ± 25 ppm) [5]. The I- of SB1-P cells removed from the liquid medium spread as a thin layer in a Petri dish and analyzed, was 91 _+ 6 ppm COs. This value was also virtually the same as the [- for SB-P cells removed from the medium (90 _+ 15 ppm) indicating that presence of the liquid medium has a comparable effect on the photosynthetic efficiency of both lines. Neither line will grow in air, however, so net photosynthesis over the entire culture period is inadequate to support cell growth and maintenance under such conditions. Measurements of RuBPcase activity document that there are only small changes in activity during the growth period. RuBPcase initial (in vivo, about 25--35 ~mol CO 2 mg -1 Chl h -1) and total (that which is present after preincubation with COs and Mg 2÷to fully activate the enzyme, about 50--80 pmol COs mg -1 Chl h -i) were quite comparable to those in SB-P cells through the culture cycle where respective initial and total rates of about 40 and 80 pmol mg -~ CO2 Chl h -1 were found. In comparison to the activities of soybean leaf RuBPcase (initial and total rates of 392 and 516 ~mol CO s mg -~ Chl h -1) [5], it is evident that the low RuBPcase activities, of both soybean photoautotrophs, are apparently a major cause of the low photosynthetic capacity of the cells. PEPcase activity in SB1-P cells showed the characteristic peak in activity early in the growth period (day 4) and then declined to a uniform level. The peak activity was about 110 pmol COs m g -1 Chl h -1 while the usual level was about 40. Due to the changes in PEPcase, the ratio of RuBPcase(I)/PEPcase activity was lowest early in the growth period, about 0.3, and then increased to a maximum of about 0.85 during the latter part of growth. These ratios were slightly lower, but comparable to, the values
measured in SB-P (RuBPcase(I)/PEPcase of 0.5 -1.5). PEPcase activity, whether expressed on a Chl basis or on a soluble protein basis, was similar to the activities found in SB-P (30--90 ~mol CO2 mg -1 Chl hbl). Low CO2 had the same effect on RuBPcase activation in SB1-P cells as it did in SB-P cells. RuBPcase initial activity, as a percentage of total activity was about 40% in SB1-P cells grown in an atmosphere of 5% CO e, which was comparable to the 50% activation level in SB-P cells in 5% COe. In SB1-P cells exposed to air for 24 h, the RuBPcase was only about 20% activated in comparison to 15% activation in SB-P cells exposed to air. These findings are in agreement with the observation from SB-P cultures where the deactivation of RuBPcase at low COs (only about 14% activated) results in severely decreased photosynthetic capacity [5], which would apparently limit photosynthesis and in turn would limit the cells ability to grow in air. Conclusions Until now no one has compared the photosynthetic characteristics of different photoautotrophic cell lines derived from the same plant species. For this purpose a second photoautotrophic soybean line, SB1-P, was established, characterized and compared to the original photoautotrophic soybean line SB-P. This study allowed us to determine if the unusual photosynthetic characteristics of SB-P in comparison to mature leaves, would be present in other soybean photoautotrophic lines, or if such characteristics were unique to SB-P. Analysis showed no significant differences in the characteristics of SB1-P compared to SB-P. Grown photoautotrophically in an atmosphere of 5% C02, SB1-P has comparable Chl levels, cell size, cell aggregate size, chromosome number, rates of growth, net photosynthetic CO2 fixation and oxygen evolution, light-dependent and dark 14C02 fixation, dark respiration and oxygen inhibition of photosynthesis. SB1-P is comparable to SB-P with regard to percent
74
RuBPcase activation, low RuBPcase activity, and PEPcase activity. Both lines are unable to grow at air levels of CO2, have high respiration, an elevated C02 compensation concentration, and show deactivation of RuBPcase at ambient levels of CO2. We conclude that SB-P and SB1-P are very similar physiologically. From the results of this investigation it is concluded that many of the unusual characteristics of these lines are the result of the conditions employed in culture, and are not due to the use of any specific soybean genotype. Studies with soybean plants have shown that decreasing the light intensity to 20% full sunlight decreased the activation state of RuBPcase from 94% to 74% [12] and increasing the CO2 level to 800 ~lfl also decreased the RuBPcase activity [13]. The photoautotrophic cultures are also grown under continuous light conditions to attempt to maximize photosynthesis unlike plants which normally undergo light and dark periods. However, when the SB-P cells were grown with a 10-h dark period, the cell growth slowed and they died within a few weeks (M. Horn, B. Martin and J.M. Widholm, unpublished). The physiology of rapidly growing photoautotrophic cells would be expected to be comparable to that of developing leaf cells. This is indicated by the active cell division, high dark respiration, low RuBPcase activity, elevated [-, and low photosynthetic efficiency, which are characteristics common between SB1-P and SB-P.
References 1
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Acknowledgements Supported in part by funds from the Illinois Agricultural Experimental Station, Agrigenetics and by the McKnight Interdisciplinary Photosynthesis Research Program.
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