Ribulose Bisphosphate Carboxylase/Oxygenase Content Determined with [<sup>14</sup>C]Carboxypentitol Bisphosphate in Plants and Algae

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Changes of some photosynthetic properties of high-CO2 grown cells of Chlorella pyrenoidosa during adaptation to low-CO2 conditions have been investigated. The K,,, value of photosynthesis of the high-CO2 grown cells for dissolved inorganic carbon was 3.3 millimolar and decreased to 25 to 30 micromolar within 4 hours after transferring to air. In the presence of saturating CO2 concentrations the photosynthetic activity of the high-CO2 grown cells was 1.5 times as high as that of the low-CO2 grown cells. There was a significant rise of the photosynthetic activity during adaptation of the high-CO2 grown cells to air, followed by a steady decrease. The activity of ribulose 1,5-bisphosphate carboxylase/oxygenase in both the high-and low-CO2 grown cells was close to the photosynthetic activity of the cells. The concentration of ribulose 1,5-bisphosphate (RuBP) was higher in the low-CO2 adapting and low-CO2 grown cells than in the high-CO2 grown cells regardless of the photosynthetic rate.This seems to be due to an increased RuBP regeneration activity during adaptation followed by maintenance of the new higher concentration. The RuBP level always exceeded the concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase RuBP binding sites in both the high-and low-CO2 grown cells at any dissolved inorganic carbon concentration.Rubisco at low CO2 and was decreased to or below the level of the binding sites with increasing atmospheric CO2 concentration. Perchorowicz et al. (17), however, obtained results with wheat leaves that were not consistent with Farquhar's model.A mechanism which seems to regulate the CO2 conditions in the cells occurs in green and blue-green algae (2,11,14,19). Algal cells grown with high-CO2 concentrations seem to lack an effective CO2 concentrating system, but the mechanism to concentrate CO2 in algal cells is induced when they are grown on low-CO2 concentrations such as air (12,13,15,18,20). The adaptation to low-CO2 conditions is completed a few hours after transferring the cells, grown on high-CO2 concentrations, to air, and is suppressed by protein synthesis inhibitors (12, 13). While several studies report the changes in photosynthetic characteristics (12,13,15,19,21) or changes in carbonic anhydrase levels and Rubisco content (8,18,20,21,23,25) that occur when cells are transferred from high-CO2 to low-CO2 conditions, there has been little information on the regulation of the photosynthetic carbon reduction cycle during this adaptation.In the present paper, we determined Rubisco content, RuBP and PGA concentrations, and photosynthetic activity during adaptation of high-CO2 grown cells of Chlorella pyrenoidosa to growth on air. Based on these results and the apparent ability for RuBP regeneration in high-and low-CO2 grown cells of Chlorella during photosynthesis, some comments are offered on the regulation of photosynthesis in Chlorella. To

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confidence: 57%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Changes of Ribulose Bisphosphate Carboxylase/Oxygenase Content, Ribulose Bisphosphate Concentration, and Photosynthetic Activity during Adaptation of High-CO₂ Grown Cells to Low-CO₂ Conditions in Chlorella pyrenoidosa

Yokota

Canvin²

1986

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“…In normal higher plants, the ratelimiting step of photosynthesis is not RuBPC, under the best conditions at least, while in green algae, one of the rate-limiting steps is this enzyme (21). Since the effect of Cbl supplement on the photosynthetic activity of the Cbl-limited cells is similar to the methionine supplementation, reduction of PSI activity may be due to a deficiency of methionine in the chloroplasts, which is synthesized by a Cbl-dependent system.…”

Section: Resultsmentioning

confidence: 98%

Photosynthesis of Euglena gracilis under Cobalamin-Sufficient and -Limited Growing Conditions

Isegawa¹,

Nakano²,

Kitaoka³

1987

Subeeilular Fractionation and Pumification of Chloroplasts. Partial trypsin digestion ofthe pellicle followed by mild mechanical disruption of Euglena ceUs and subcellular frationation by differential centifugation were conducted according to Shigeoka et al. (19).A suspension of chloroplasts was layered on the top of continuous Percoll (Lot No. 4634) gradients and centrifuged in a swingout rotor (Hitachi Reactions of Photosynthesis. Photosynthesis resulted in net 02 evolution from cells, which were cultured in the dark in the presence of Cbl and then in the light with limiting or supplementing CbL was measured with a Kalbani-type oxygen electrode (Kyusui Kaaku Co.) in 2 ml of a reaction mixture containing an aliquot ofthe cell culture and 25 mm NaHCO3, 1 mM MgC12, and 50 mmt potassium phosphate buffer (pH 7.8). The mixture which contained 60 to 100 ug Chl ml-', was incubated at 25°C and 10,000 lux with wolfram lamp. 4C02 fixation was carried out under the same conditions as described above except that 10 mm (0.4 MCi) (final concentration) NaH'4C03 was added instead of 25 mm NaHCO3. A sample of 0.2 ml was withdrawn after a definite period through Eppendorf pipette and injected into a vial containing 0.3 ml of 0.3 N HCI. The vial was shaken for 30 mn to remove free`4C02 and the fixed radioactivity in the sample was counted with Aloka LSC 903 scintillation counter.

“…During the C02-free treatment with maize, RuBP was increased up to 6-fold, but when CO2 was reintroduced RuBP quickly returned, indicative that the pool size was not as important as its rate of turnover. (4,23) and it will displace any other known inhibitor (19).…”

Section: Discussionmentioning

confidence: 99%

Photosynthesis and Activity of Ribulose Bisphosphate Carboxylase of Wheat and Maize Seedlings during and following Exposure to O₂-Low, CO₂-Free N₂

Gustafson¹,

Raynes²,

Jensen³

1987

Seven day old wheat and maize seedlings were exposed to 1300 or 2000 microeinsteins per square meter per second photosynthetically active radiation in CO2-free air for 3 hours with either 1% 02 in N2 or N2-only and then returned to normal air of 340 microliters per liter C02, 21% 02 in N2. Activity of the ribulose bisphosphate carboxylase and amount of the substrate, ribulose 1,5-bisphosphate, were measured during and following the C02-free treatments as was photosynthetic CO2 fixation. Photoinhibition of photosynthesis was observed only with wheat seedlings following the N2 only treatment. During the C02-free treatments, the levels of RuBP rose during all experiments except when wheat was photoinhibited. The activity of the ribulose bisphophate carboxylase, measured directly upon grinding the leaves, declined during the CO2-free conditions. The carboxylase total activity increased in minutes in the leaf during and following the CO2-free treatments. cient 02 is present to support photorespiration (15). We have examined effects on the activity of Rubisco and on leaf RuBP levels under conditions reported to cause photoinhibition. Wheat and maize seedings, 7 d old, were illuminated in CO2-free atmospheres for 3 h. To our surprise, photoinhibition was induced only in wheat when the seedlings were exposed to high light with N2 only, free of CO2 and 02. Although Rubisco is partially deactivated in C02-free air, the major cause of photoinhibition of CO2 fixation was, most likely, the impaired ability of the photosystems to support RuBP regeneration rather than deactivation of Rubisco.Our earlier measurements of Rubisco activation were reported as percent activation, which was the ratio of the initial activity to the total activity times 100 (1 1, 12). Initial activity refers to the CO2 fixation activity of Rubisco in an extract, measured directly following grinding the leaf at 0C with rate-saturating levels of RuBP, C02, and Mg2' at 25°C. The Rubisco total activity is the CO2 fixation activity following full activation of Rubisco in CO2 and Mg2e for 10 min. The initial activity of Rubisco from wheat seedlings exposed to N2-only (no 02) was found to be near zero while the RuBP concentrations were at the apparent binding site levels of the enzyme (11). In those experiments we did not observe much change in the total activity. Recent experiments, especially under low CO2 and 02 levels, indicates that the total activity can change substantially. MATERIALS AND METHODSLittle is known of the response of Rubisco2 activity in leaves exposed to C02-free atmospheres. The ability of 02 to support activation of Rubisco in the absence of CO2 was reported by Perchorowicz and Jensen (11). They suggested that 02 was involved in activation by turning over RuBP during photorespiration to form PGA and P-glycolate. This would cycle the NADPH and ATP and thereby maintain electron flow through the photosystems. Photorespiration could also supply cellular levels of CO2 needed to support activation of Rubisco (5).Illumination Gas excha...