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Regulation of Ribulose-1, 5-Bisphosphate Carboxylase

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Regulation of Ribulose-1, 5-Bisphosphate Carboxylase Plant Physiol. (1993) 102: 21-28 Regulation of Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase Activity in Response to Reduced Light lntensity in C4 Plants' Rowan F. Sage* and Jeffrey R. Seemann Department of Botany, University of Georgia, Athens, Ceorgia 30602 (R.F.S.); and Department of Biochemistry, University of Nevada, Reno, Nevada 89557 (J.R.S.) tion of Rubisco activity by reversible carbamylation occurs in lhe light-dependent regulation of ribulose-1,5-bisphosphate response to changes in light intensity as well as the concen- carboxylase/oxygenase (Rubisco) activity was studied in 16 species tration of C02and 02,whereas inhibition of Rubisco activity of C, plants representing all three biochemical subtypes and a by CAlP occurs only in response to varying PPFD (Sharkey variety of taxonomic groups. Rubisco regulation was assessed by et al., 1986; Sage et al., 1990; Seemann et al., 1990). At measuring (a) the ratio of initial to total Rubisco activity, which physiological levels of COz in C3 plants (5-10 PM), full reflects primarily the carbamylation state of the enzyme, and (b) carbamylation of Rubisco requires Rubisco activase (Salvucci, total Rubisco activity per mo1 of Rubisco catalytic sites, which 1989; Portis, 1990). In the absence of Rubisco activase, only declines when 2-carboxyarabinitol I-phosphate (CAlP) binds to carbamylated Rubisco. In all species examined, the activity ratio of 20 to 40% of Rubisco catalytic sites are carbamylated under Rubisco declined with a reduction in light intensity, although sub- physiological conditions, leading to a significant inhibition of stantial variation was apparent between species in the degree of photosynthesis (Salvucci, 1989; Portis, 1990). However, full Rubisco deactivation. No relationship existed between the degree carbamylation of Rubisco can occur if COz levels are in- of Rubisco deactivation and C, subtype. Dicots generally deacti- creased well above ambient in the absence of RuBP (above vated Rubisco to a greater degree than monocots. lhe total activity 100 WM; Andrews and Lorimer, 1987). of Rubisco per catalytic site was generally independent of light In C4 plants, the C02-concentrating mechanism is esti- intensity, indicating that CAlP and other inhibitors are not major mated to increase the COz concentration in the bundle sheath contributors to the light-dependent regulation of Rubisco activity 10- to 100-fold above that in the mesophyll tissue (Furbank in C., plants. lhe light response of the activity ratio of Rubisco was measured in detail in Amaranthus retroflexus, Brachiaria texana, and Hatch, 1987; Jenkins et al., 1989). This high COz partia1 and Zea mays. In A. retroflexus and 6. texana, the activity ratio pressure may promote full carbamylation of Rubisco, even in declined dramatically below a light intensity of 400 to 500 pmol of the absence of Rubisco activase, raising the possibility that photons m-z s-'. In 2. mays, the activity ratio of Rubisco was reversible carbamylation is not an important regulatory relatively insensitive to light intensity compared with the other mechanism in C4 plants. Results of a previous study of the species. In A. retroflerus, the pool size of ribulose bisphosphate regulation of Rubisco activity in the C4 plant Zea mays support (RuBP) declined with reduced light intensity except between 50 this hypothesis, because the degree of Rubisco deactivation and 500 pmol m-* s-', when the activity ratio of Rubisco was light in darkness is much less in Z. mays than in C3 plants (Usuda, dependent. In Z. mays, by contrast, the pool size of RuBP was light 1990). However, in the presence of RuBP and absence of dependent only below pmol m-* s-'. lhese results indicate 350 Rubisco activase, carbamylation of Rubisco from C3plants is that, in response to changes in light intensity, most C, species inhibited, even at high concentrations of C02 (Portis et al., regulate Rubisco by reversible carbamylation of catalytic sites, as commonly observed in C3 plants. In a few species, notably Z. mays, 1986; Robinson et al., 1988). Consequently, Rubisco activase Rubisco is not extensively regulated in response to changes in light may be required by C4 plants to fully carbamylate Rubisco. intensity, possibly because the activity of the COz pump may C4 plants are known to contain Rubisco activase (Salvucci et become limiting for photosynthesis at subsaturating light intensity. al., 1987). Rubisco activity in C4 plants may also be inhibited by light-dependent binding of CAlP, because CAlP occurs in darkened leaves of some C4 species (Moore et al., 1991), In C3 plants, the activity of Rubisco is postulated to be and Rubisco activity in darkened leaves of a few C4 species regulated either by reversible carbamylation of a Lys residue is inhibited by a tight binding inhibitor, putatively CAlP (Vu in the catalytic site, enabling catalysis, or by the binding of et al., 1984; Servaites et al., 1986). inhibitors such as CAlP to carbamylated catalytic sites, dis- To understand better the regulation of Rubisco in Cqplants, abling catalysis (Seemann et al., 1990; Portis, 1992). Regula- we studied the light-dependent regulation of Rubisco activity in 16 C4 species representing each biochemical subtype ' Research supported by National Science Foundation grant 8906390 to R.F.S., a fellowship to R.F.S. from the Sarah Moss Abbreviations: CAlP, 2-carboxyarabinitol l -phosphate; CABP, 2- Foundation, and U.S.Department of Agriculture-National Research carboxyarabinitol1,5-bisphosphate;NAD(P)-ME, NAD(P)-malic en- Initiative grant 91-37306-6474 to J.R.S. zyme; PCK, phosphoenolpyruvate carboxykinase; PGA, 3-phospho- * Corresponding author; fax 1-404-542-1805. glyceric acid; RuBP, ribulose 1,5-bisphosphate. " LI 22 Sage and Seemann Plant Physiol. Vol. 102, 1993 (NADP-ME, NAD-ME, and PCK) and a variety of taxonomic inhibition (Seemann et al., 1985; Kobza and Seemann, 1988; groups. Seemann et al., 1990). Leaves were extracted at O to 4OC in a pH 8.0 buffer MATERIALS AND METHODS containing 100 mM Bicine, 1 mM DTT, 1 mM Na-EDTA, 3.3% (w/v) insoluble PVP, 0.14% (w/v) BSA, 20 mM MgC12, and Plant Material 0.15 p~ NaHC03. This extraction buffer contains more ' The effect of light intensity on the activity of Rubisco was NaHC03 and MgC12 than used previously (Sharkey et al., examined in 16 species of C4 plants, one C3-C4intermediate 1986; Sage et al., 1990) because it better maintained the species, and four C3 species. Plants were grown in either an activity of Rubisco in initial extracts (data not presented). A outdoor garden, a greenhouse, or natural stands in the field 100-pL aliquot of the extract was immediately assayed (see Table I for species list and associated growth conditions). (within 150 s of extraction) by adding it to 400 pL of assay Plants were selected to represent each C4 subtype, including buffer (100 mM Bicine [pH 8.21, 1 mM Na-EDTA, 0.1-0.3 both monocots and dicots. The c3-C~intermediate and C3 units mL-' of ribulose-5-P kinase, 1 unit mL-' phospho- plants were included in the study for comparative purposes. ribuloisomerase, 2.0 mM ATP, 1.6 mM ribose-5-P, 28 mM Five species from the genus Panicum, three C4 species repre- MgC12, and 19 mM ['4C]NaHC03; specific radioactivity of senting each subtype, one C3-C4 intermediate species, and 20.2 Bq nmol-'), which was modified from that used by one C3 species, were also included. Complete light responses Seemann and Sharkey (1986). RuBP was generated in situ of Rubisco regulation were determined for three species, from ribose-5-P using ATP, ribulose-5-P kinase, and phos- Amaranthus retroflexus L. (NAD-ME type), Zea mays L. phoribuloisomerase. After a 30-s assay, the reaction was (NADP-ME type), and Brachiaria texana (Buckley) S.T. Blake terminated with 2 N HCl, and the acid-stable radioactivity (PCK type). was determined by liquid scintillation spectroscopy. Plants in the greenhouse were grown in a 6-L pot contain- Immediately after the initial activity was assayed, a 900- ing a peat:perlite loam mixture (Fafard mix No. 3; Conrad pL aliquot of crude extract was treated with 100 pL of an Fafard Inc., Springfield, MA) and were fertilized daily with activation buffer (100 mM MgC12, 200 mM NaHC03, 100 mM a Johnson-Hoagland solution (Epstein, 1972) modified to Bicine [pH 8.21) to activate Rubisco. After the aliquot was contain 20 FM iron as Sequestrene 138 (Ciba-Geigy Corp., incubated 10 to 16 min at room temperature, Rubisco activity Greensboro, NC). Plants grown in an outdoor garden were was assayed as above to give the activity of fully carbamy- imgated and fertilized with the modified Johnson-Hoagland lated Rubisco. Rubisco content in 60 to 90 pL of the activated solution as necessary to avoid signs of water and nutrient extract was then assayed by determining the amount of stress. Plants from natural stands received no care. [14C]CABPbound to Rubisco, as described previously (See- Light treatments were established by shading the plants mann and Sharkey, 1986). with plastic netting of neutra1 density and/or cloth fabric. The carbamylation state of Rubisco was also assessed using After a minimum of 60 min at a given light intensity, ap- a CABP-trapping assay modified from Butz and Sharkey proximately 4 cm2 of leaves were sampled with a portable, (1989). Samples were extracted in the same extraction buffer hand-operated freeze-clamp device. The copper clamping described above for activity assays. Immediately after the heads were prechilled in liquid nitrogen. Samples were stored extract was centrifuged, a 60-pL aliquot was added to 400- in liquid nitrogen until assayed. Samples were collected be- pL microcentrifuge tubes containing 100 pL of the extraction tween 10 AM and 4 PM at temperatures ranging between 22 buffer and 2 pL of 2 mM [14C]CABP(with some carboxyribitol and 35OC.
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