Orsay Cedex, France (B.N-Q.,M.K.,A.L.); and U.S. Department of Agriculture, Agricultural ... the young leaf showed a high sucrose synthase (SS) activity. Leaf SS ...
Received for publication March 27, 1990 Accepted May 24, 1990
Plant Physiol. (1990) 94, 516-523 0032-0889/90/94/051 6/08/$01 .00/0
Sucrose Synthase in Developing Maize Leaves Regulation of Activity by Protein Level during the Import to Export Transition Binh Nguyen-Quoc1, Micheline Krivitzky, Steven C. Huber, and Alain Lecharny* Laboratoire de Structure et Mtabolisme des Plantes, CNRS (URA 1128), Universit6 de Paris-Sud, BAt. 430, 91405 Orsay Cedex, France (B.N-Q.,M.K.,A.L.); and U.S. Department of Agriculture, Agricultural Research Service, and Department of Crop Science and Botany, North Carolina State University, Raleigh, North Carolina 27695-7831 (S.C.H.). glucosyltransferase EC 2.4.1.14) catalyzes a reaction which may be rate limiting for sucrose synthesis (10, 15, 20). During spinach leaf development, increased SPS activity followed closely the increase in enzyme-protein level (25). A sink leaf tissue is a sucrose utilization sink in which hexose-P are obtained from the sucrolytic breakdown of imported sucrose. It is generally considered that the sucrose gradient necessary to sustain the phloem unloading is, in part, controlled by the utilization of sucrose in the cytoplasm of the sink cells. Two enzymes may cleave imported sucrose: invertase (f,-D-fructofuranoside fructohydrolase, EC 3.2.1.26) which catalyzes the cleavage of sucrose to glucose + fructose and sucrose synthase (UDP-glucose: D-fructose 2-glucosyltransferase, EC 2.4.1.13) which catalyzes the reversible interconversion of sucrose and UDP to UDP-Glc + Fru. The sucrose synthase pathway for sucrose breakdown has been described recently (1, 13). Activities in extracts from young leaves suggest that either invertase or SS may have the major role in sucrose cleavage depending on the plant examined (24). Both acid invertase activity (11) and SS activity (3, 14, 21) decline during leaf expansion. Schmalstig and Hitz (22) estimated the relative in vivo activities ofinvertase and SS in expanding soybean leaves. In very young leaves, all sucrose cleavage was by SS. Furthermore, sucrose cleavage activities through the SS pathway apparently reflect the sink strength in certain plant sinks (6, 18, 23). In developing maize kernels (5, 8, 16), two sucrose synthase isozymes, SS, and SS2, are encoded by separate genes, Shrunken and Sus respectively, both located on chromosome 9 (4). While expression of both SS genes exhibits a complex tissue and temporal specificity (2, 9) the two izozymes are thought to have the same physiological function, i.e. sucrose hydrolysis. Our preliminary experiments confirmed that SS activity was high in heterotrophic maize leaf tissues and almost not detectable in mature green tissues. To get more information on the role of SS in maize leaves we studied the changes in SS activity during the import to export transition. Changes in the Chl content and in SPS activities were taken as an indication respectively of changes in photosynthetic and export capacities of the tissues. During leaf development there was a rapid drop of SS activity before SPS activity increased. Clearly, sucrose cleavage through SS was down regulated before sucrose synthesis was initiated. Changes in SS activity
ABSTRACT The maize (Zea mays) leaf is a valuable system to study the sucrose import to sucrose export transition at the cellular level. Rapidly growing and fully heterotrophic cells in the basal part of the young leaf showed a high sucrose synthase (SS) activity. Leaf SS has been purified to homogeneity. By comparison with purified kernel SS isozymes, the leaf SS has been identified as SS2. SS, protein and SS2 protein were clearly separated by electrophoresis and the two monomers differed in size by 6 kilodaltons. Nevertheless, kinetic parameters of both enzymes were very similar. Immunodetection of SS protein showed that in young heterotrophic tissues SS2 was a major protein accounting for 3% of the total protein. Concurrent with greening, SS activity decreased and the change of activity was explained by regulation of the protein level. In mature green tissues, which are synthetizing sucrose as evidenced by the presence of sucrose phosphate synthase activity, SS activity was almost completely absent. Results suggested that down regulation of SS2 enzyme protein level was an early event in the transition from import to export status of the leaf.
During leaf development, potentially photosynthetic leaf cells progressively acquire their photosynthetic capacity by differentiation of proplastids into functional chloroplasts. Simultaneously, and at an appropriate time, the sucrose import process must be turned off while the sucrose synthesis and the export start to function. While the developmental and environmental control of chloroplast gene expression during greening has been extensively studied (see the recent review by Gruissen [7]), the cellular mechanisms underlying the transition from sucrose import to sucrose export has received little attention (24). Analysis of events involved in this metabolic reorientation is now possible since the likely key enzymes catalyzing rate limiting steps in importing and exporting cells have been identified in the last few years. Assimilate export rate depends on the flow rate of carbon through the sucrose synthesis pathway (19). There are indications that SPS2 (UDP-glucose: D-fructose 6 phosphate 2' Recipient of a fellowship from the Government of France (Ministere des Affaires Etrangeres). 2 Abbreviations: SPS, sucrose phosphate synthase; FL, full length; SS, sucrose synthase.
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2 min at 12,000 g. Protein determination (Bio-Rad assay) was made on the supernatant. The supernatants were desalted by centrifugation on Sephadex G 25 (Pharmacia Fine Chemicals) columns equilibrated in buffer B (50 mm Hepes/NaOH [pH 7.5], 2 mM MgCl2, 1 mM EDTA, 2 mM DTT) plus 0.1% BSA. Recoveries of SS protein, estimated by immunorocket analysis, were always more than 85% and SS activities were corrected for recovery of SS protein. Enzymes were always assayed 30 min after extraction.
were mainly explained by a control of the enzyme-protein level. These results prompted us to further characterize SS from maize leaves and to compare it with the kernel isozymes. MATERIALS AND METHODS Plants
Zea mays L., genotype F7F2 was grown in a greenhouse in plastic pots filled with vermiculite. Germination conditions and nutrient solution were as described elsewhere (19). Minimal day/night temperatures were 25°C/ 1 8°C, RH was 50 to 60%, natural light was supplemented 16 h each day with fluorescent lamps (Phytoclaude, 200 ,umol quanta m-2 s-'). At the time the fourth leaf from the base was just emerging from the third leaf, plants were transferred to continuous light (200 Amol quanta m-2 s-') and sampled every 6 h (first experiment) or every 24 h (second experiment). The fourth leaf was excised at the level of the ligule of the first leaf; three parts (each 3 cm long) were cut from the tip, the middle, and the base of the leaf. Each time samples were taken from six plants, arranged in two groups for analysis, rapidly weighed, deep frozen in liquid nitrogen, and kept at -80°C before extraction. Differences (15-50%) were observed in growth rate and the other parameters measured between experiments but the general patterns were not altered. An adjusting factor was computed from the ratio of(first experiment data)/second experiment data) and applied to the data from experiment 2.
Assays for Enzymes
Except when Vmax and Km values for SS were determined, enzymes were assayed under Vmax substrate concentrations, at 30°C, in a total volume of 1 mL. SS in the breakdown direction was assayed as previously described (13); 100 mM sucrose, 1 mM ATP, 0.4 mM NAD were added to buffer B at 30°C with four units of phosphoglucoisomerase, four units of hexokinase, and two units of Leuconostoc glucose-6-P dehydrogenase as coupling enzymes. Invertase activity was calculated from the A340 increase and then 1 mm UDP was added to start the SS reaction. SPS activities were determined by a method derived from Huber et al. (12) in the presence of 10 mM Fru 6-P, 40 mM Glc 6-P, and 10 mM UDPGlc in buffer B. The fructose moeity of sucrose-P + sucrose was determined by the resorcinol method. SS Purification
Extraction
SS was purified both from whole developing kernels harvested 30 d after pollination and from basal parts of leaves from 15 d old plants. The same steps were used. Fifty grams fresh weight were ground in a mortar with liquid nitrogen and suspended in 100 mL of extraction buffer A containing the protease inhibitors PMFS (1 mM) and leupeptin (10 ,M). The
Leaf samples were ground in a mortar with liquid nitrogen and suspended, at 4°C, in buffer A (50 mM Hepes-NaOH [pH 7.0], 10 mM MgC12, 1 mM EDTA, 2 mM DTT, 10% [v/v] ethyleneglycol, 0.02% [v/v] Triton X-100). An aliquot was retained for Chl assay. The homogenate was centrifuged for I
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Figure 1. Changes in (A) length, (B) Chi content, and (C) protein content in the fourth leaf of maize plants in continuous light. A, Different symbols for leaf length are from two independent experiments. B and C, (0) basal part of the leaf; (*), middle part of the leaf; (U), tip of the leaf.
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NGUYEN-QUOC ET AL.
homogenate was filtered and precipitated with PEG (mol wt, 8000) between 9 and 14% (v/v). The 14% pellet was resuspended with 10 mL buffer A and loaded at 10 mL h-' onto an UDPGIcUA-agarose affinity column (Sigma) of 2 mL equilibrated with buffer B. After washing with 100 mL of buffer B, SS was eluted with 10 mL of 150 mM KCl in buffer B. This fraction was added to 30 mL of buffer C (50 mM Hepes/NaOH [pH 7.5], 10 mM MgCl2, 2 mM EDTA), loaded onto a 1 mL mono-Q column (Pharmacia) equilibrated with buffer C and eluted with a linear gradient of KCI, 0 to 400 mm, in buffer C. Fractions containing the highest SS activity were pooled and desalted-concentrated by centrifugation onto a centricon 30 membrane (Amicon). Pure SS was stored at -20°C in 20% (v/v) glycerol or in 1% (w/v) BSA depending on its future use. Fifteen percent of activity was lost after 6 weeks.
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apoferritin, gammaglobulin, aldolase, ovalbumin, myoglobin, and vitamin B 13 as calibration proteins.
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