Role of the pentose phosphate pathway during callus development in explants from potato tuber

Plant & Cell Physiol. 12: 73-79 (1971) Role of the pentose phosphate pathway during callus development in explants from potato tuber YOSHIO KIKUTA, TETSUO AKEMINE and TAKASHI TAGAWA Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo, Japan (Received October 15, 1969) Metabolic change during callus formation in explants from potato tuber (Solatium tuberosum L.) was investigated. Under the present culture conditions, callus starts exponential growth (estimated by the fresh weight increase) with a 5-day lag period, at which time marked production of DNA occurs. In the lag period, respiration is very much resistant to malonate, and the value of the C6/C1 ratio and activities of both G6PDH 6PGDH pass their peaks. On the basis of these and relevant findings, glucose metabolism through the PP pathway is surmised to play a significant role in the initial DNA multiplication phase of callus development. Callus formation from explants of potato tuber has been reported (1). Exogenous application of auxin is indispensable for initiating callus, but no additional application of kinetin is required (2). Respiratory and glucose metabolisms of cultured carrot root tissues showed remarkable increases in activity of the glycolytic- TCA pathway during the first 4 6 days of culture and the predominance of the PP pathway during callus development (3). Potato tissues metabolize glucose via the PP pathway under certain physiological conditions (4). The present experiments suggest a role of the PP pathway in the initial phase of callus development in isolated potato tuber tissues in vitro. Materials and methods Plant materials and tissue culture: Discs, 1.0 mm thick and 5.0 mm in diameter, were prepared from the tuber of Solanum tuberosum L., var. Irish Gobbler, and were aseptically grown on 30 ml of modified White's medium in 100 ml Erlenmeyer flasks in a dark room at 25 C for 3 weeks (/). NAA (1.6X10" 6 M) was added to the medium to stimulate callus development. At appropriate intervals aliquots of the explants were harvested for analyses as described below. Analytical procedures: Harvested explants were washed with distilled water and blotted dry with filter Abbreviations: PP pathway, pentose phosphate pathway; TCA cycle, tricarboxylic acid cycle; G6PDH,rf-glucose-6-phosphate : NADP oxidoreductase; 6PGDH, 6-phospho-rf-gluconate : NADP oxidoreductase (decarboxylating); NAA, a-naphthaleneacetic acid. 73

74 Y. KIKUTA, T. AKEMINE and T. TAGAWA paper, then their fresh weights were determined. Approximately 1.0 g of explants, as intact blocks, was placed in the main compartment of a Warburg respirometer. One ml of 0.05 M phosphate buffer, ph 5.6, containing 3 /id of glucose-u- 14 C (30 mci/mmole), or 1.0 /*Ci of glucose-l- I4 C or glucose-6-14 C of equal specific activities (10 mci/mmole) was placed in the side arm and 0.3 ml of 5 N K.OH was placed in the center well. In some experiments malonate was also placed in the side arm at a final concentration of 5x 10" 2 M. The solution in the side arm was poured into the main compartment at zero time after thermal equilibration at 25 C. Evolution of 14 COz from labeled glucose was determined by the technique of ap Rees and Beevers (4). After explants were exposed to labeled glucose for 3 hr, they were collected and rinsed with non-radioactive 0.05 M phosphate buffer, ph 5.6, then were immediately crushed with 80% methanol in a mortar. Homogenates were successively treated with absolute methanol, ether and acetone-ether (2:1, v/v). The resulting dry powder was resuspended in 5 ml of 5% trichloroacetic acid at 0 C and centrifuged at 5,000 x^ for 5 min to obtain the acid soluble fraction. The RNA fraction was extracted with 5 ml of 2.0 N perchloric acid at 2 C overnight, then rinsed with cold acid. Precipitates were extracted with 4 ml of 0.5 N perchloric acid at 90 G for 30 min to obtain the DNA fraction. Nucleic acids were determined by the method of Ogur and Rosen (5). The residue was solubilized with 5 ml of 2.0 N NaOH 37 C overnight. Aliquots of the five fractions (methanol-acetone-ether, acid soluble, RNA, DNA and residual fractions) were spread on stainless steel planchets and dried, then was assayed for radioactivity. Glucose uptake was determined by summing up radioactivities of the five fractions and that of the respiratory 14 CO 2 fraction. For counting "C-RNA, aliquots of the RNA fraction were hydrolyzed in 0.3 N KOH at 37 C for 18 hr. Resulting nucleotides and contaminated glucose were separated by paper chromatography using water as the developing solvent. Spots detected in UV light were counted. Glucose-U- 14 C, glucose-l- 14 G and glucose-6-14 C were obtained from the Radiochemical Centre, Amersham, Buckinghamshire England. G6PDH and 6PGDH activities were assayed as follows. Twenty explants were ground in a mortar with 10 ml of a grinding solution containing 0.3 M sucrose, 0.005 M cysteine and 0.05 M Tris buffer, ph 8.0. Homogenates were centrifuged at 15,000 Xg for 30 min, then the supernatants were dialyzed overnight against 1.0 liter of 0.02 M Tris buffer, ph 7.0. The above procedures were carried out at 2 C. The volume of dialyzed solution was adjusted to 30 ml with 0.05 M Tris buffer, ph 7.0. The reaction mixture for the enzyme assay contained 1.0 ml of enzyme solution, 10/jmoles of MnSC>4, 340 /jmoles of Tris buffer, ph 7.0 and 1.0/tmole of substrate (glucose-6-phosphate or 6-phosphogluconate) and was made up to 2.9 ml with water. The reaction was initiated by adding 0.1 ml of 0.005 M NADP. Enzymic activity was measured at 25 G by recording the absorbance change at 340 run. Specific activity was defined as the number of /^moles of NADPH 2 produced per min per mg protein. Protein was precipitated with 10% trichloroacetic acid, then determined by nesslerization. Glucose-6-phosphate, 6-phosphogluconate and NADP were purchased from Sigma Chemicals Co., St. Louis, Missouri U. S. A.

Pentose pathway in potato tissue cultures 75 Results Fig. 1 shows growth (fresh weight increase) of explants in the presence and absence of NAA and changes in the fresh weight/dna ratio in the presence of added NAA. When NAA was absent, explants remained alive during 3 weeks' culture period with no sign of growth or callus formation. In the medium with added NAA, callus formed from explants and grew exponentially with an initial lag period of 5 days. The maximal growth rate was attained 8-15 days after culture started. Since the fresh weight/dna ratio was markedly reduced, first in the initial lag phase and then in the exponential growth phase, this may reflect stimulated DNA synthesis accompanied by no and active cell division and expansion, respectively. Changes in the rate of respiration and the rate of glucose uptake in explants cultured with or without added NAA are shown in Fig. 2 and 3, respectively. In the presence of added NAA, the O2 and glucose uptake rates increased 2 and 4 fold, respectively, during the initial 24 hr period of culture. Similar but slightly less significant changes were observed in the absence of added NAA. Significantly, only when NAA was added were these activities per explant once more elevated Fig. 1. Callus development in explants from potato tuber. Three explants (1x5 mm, disc) were aseptically cultured in 30 ml of modified White's medium in a 100 ml Erlenmeyer flask at 25 C in the dark. +NAA and NAA indicate the presence and absence, respectively, of 1.6 X 10" 6 M NAA in the medium. DNA was estimated by the method of Ogur and Rosen (5). 10 15 Time, Ooys 20 Fig. 2. Change in the rate of oxygen uptake during callus development in explants from potato tuber. +NAA and NAA indicate the presence and absence of 1.6 X 10" 6 M NAA in the medium. Culture conditions were the same as in Fig. 1. 10 15 20

76 Y. KIKUTA, T. AKEMINE and T. TAGAWA 10 15 5 10 15 20 5 10 15 20 Fig. 3. Change in the rate of glucose uptake by explants from potato tuber. Explants cultured with or without added 1.6 X IO" 6 M NAA ( + NAA or -NAA) were harvested at appropriate intervals and exposed to 3 fid of glucose- U- U C (10-* M) at 25 C for 3 hr. Experimental procedures are detailed in Materials and methods. Fig. 4. Changes in inhibitory action of malonau on u C0z evolution from glucose-u- u C by explants from potato tuber. Explants cultured with or without added 1.6X10~ 6 M NAA (+NAA or NAA) were harvested at appropriate intervals and exposed to 3 //Ci of glucose-u- 14 C (10"* M) at 25 C for 3 hr in the presence and absence of 5 x 10" 2 M malonate. Experimental procedures are detailed in Materials and methods. Fig. 5. Change in C6/C1 ratio during callus development in explants from potato tuber. Explants cultured with or without added 1.6X10" 6 M NAA (+NAA or -NAA) were harvested at appropriate intervals and exposed to 1.0 //Ci ofglucose-l- u Corglucose-6-"C of equal specific activities (lomci/mmole) at 25 C for 3 hr. Experimental procedures are detailed in Materials and methods.

Pentose pathway in potato tissue cultures 77 Fig. 6. Clucose-U- li C incorporation into RNA and respiratory carbon dioxide in the presence of malonate in explants from potato tuber. Explants cultured with added 1.6xlO" a M NAA were harvested at appropriate intervals and exposed to 3 fid of glucose-u-'*c (10~ 4 M) in the presence of 5x 10~ 2 M malonate at 25 C for 3 hr. Experimental procedures are detailed in Materials and method. 5 10 15 20 in the exponential growth phase. No such second elevation occurred in the absence of NAA. The first rise in activities may be attributed to enhancement of activities in individual cells, while the second rise may be due to cell multiplication and growth. One marked effect of NAA was an increase in malonate insensitive decarboxylation, probably via the PP pathway during the initial lag phase of callus development, while malonate sensitive decarboxylation, probably via the TCA cycle, appeared to increase during the exponential growth phase (Fig. 4). However, in the absence of added NAA sensitivity to malonate was gradually lost after reaching a peak in the early period of culture. NAA tended to decrease the C6/G1 ratio in the initial lag phase of callus development (Fig. 5). This also supports the above conclusion of participation of the PP pathway in the initial lag phase. The rate of M CO 2 evolution from glucoes-u- 14 C in the presence of 5 X 10" 2 M malonate increased 20 fold in the initial 24 hr period of NAA culture, and showed changes parallel to these for "C incorporation into RNA during the time-course of callus development (Fig*.6). Since glucose was metabolized, probably through the PP pathway in the initial lag phase of callus development, G6PDH and 6PGDH were expected to be enhanced Culture conditions Freshly cut Control 1.6xlO" 7 M NAA 1.6X10-6 M NAA 1.6X10" S M NAA 5.3X10-5 M NAA Table 1 Activity of C6PDH and 6PGDH in explants cultured with added NAA for 24 hr G6PDH Specific Reltaive : activity " activity 239 100 255 107 322 135 358 150 304 128 362 152 Specific activity 50 97 120 129 148 6PGDH Relative activity 100 194 240 258 295 Specific activity is denned as the amount of enzyme which produces one m^mole of NADPH 2 per min per mg protein.

78 Y. KIKUTA, T. AKEMINE and T. TAGAWA 100 10 15 20 Fig. 7. Change inactivity 0JG6PDH and 6PCDH during callus development in explants from potato tuber. Explants cultured with added 1.6X10" 6 M NAA were harvested at appropriate intervals and used for enzyme assays. All assays were performed as described in Materials and methods. The maximum specific activity reached by each enzyme during the culture period was taken as 100%. in the same period of time. Table 1 shows the promotive effect of added NAA on G6PDH and 6PGDH in the initial 24 hr period of culture. Specific activity of G6PDH was unchanged in the control culture and was increased by added NAA by ca. 30-50%, while the specific activity of 6PGDH was doubled and tripled in the absence and presence of added NAA, respectively. The relative activities of G6PDH and 6PGDH in explants cultured with added NAA increased during the initial 2-day period, then gradually decreased in the subsequent period (Fig. 7). Discussion Under the present culture conditions, callus started exponential growth with an initial lag phase of 5 days during which a marked production of DNA occurred. 1. Metabolism in the initial lag phase of callus development In the initial 5-day period, respiratory activity was elevated regardless of the presence or absence of NAA in the medium. This will be related to the increase in glucose uptake which was little affected by added NAA. A marked enhancement of the PP pathway by added NAA is suggested by the fagt that "CO 2 production from glucose-u- 14 C became very much insensitive to malonate. Promoted activities of G6PDH and 6PGDH by added NAA also suggest that the PP pathway is involved in the initial lag phase of callus development. 2. Metabolism during the exponential growth phase of callus development The second rise in respiratory activity and in DNA synthesis occurred between the 8th to 15th day when callus grew exponentially; presumably with cell division and expansion. U CC>2 production from glucose-u- 14 C became much more sensitive to malonate {ca. 50% inhibition) and the C6/C1 ratio was also elevated, indicating restoration of the TCA cycle activity. 3. Role of the PP pathway during callus formation Activation of the PP pathway by added NAA may increase the amount of available precursors and NADPH 2 for syntheses of cellular components. A parallel was detected between RNA synthesis and CO2 production in the presence of malonate. Synthesis of nucleic acids and proteins, induced by added NAA, has been shown to play an important role in callus formation in potato tuber tissue (6").

Pentose pathway in potato tissue cultures 79 References ( 1) Okazawa, Y., N. Katsura and T. Tagawa: Effects of auxin and kinetin on the development and differentiation of potato tissue cultured in vitro. Physiol. Plant. 20: 862-869 (1967). (2) Okazawa, Y.: Significance of native cytokinin in callus growth of potato tissue cultures. Proc. Crop Sci. Soc. Japan 37: 522-527 (1968). ( 3) Komamine, A., Y. Morohashi and M. Shimokoriyama: Changes in respiratory metabolism in tissue cultures of carrot root. Plant & Cell Physiol. 10: 411-423 (1969). (4) ap Rees, T. and H. Beevers: Pentose phosphate pathway as a major component of induced respiration of carrot and potato slices. Plant Physiol. 35: 839-847 (1960). (5) Ogur, M. and G. Rosen: The nucleic acids of plant tissues I. The extraction and estimation of deoxypentose nucleic acid and pentose nucleic acid. Arch. Biochem. 25: 262 276 (1950). (6") Okazawa, Y.: Action of inhibitors of RNA synthesis on auxin induced callus formation of potato tissue. Proc. Crop Sci. Soc. Japan 38: 622-625 (1969).