Photosynthetic Carbon Metabolism in Isolated Maize Bundle Sheath Strands

Abstract

Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles C02 per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic "4CO2 fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath '4CO2 fixation. plants which distinguishes them from C3 species is the presence of two major chloroplast-containing leaf cell types: the bundle sheath cells which tightly surround the vascular tissue, and the mesophyll cells which in turn surround the bundle sheath layer (24).It has been suggested that in the C, panicoid grasses such as maize, sugarcane, and crabgrass, atmospheric CO2 is initially fixed by PEP carboxylase in the mesophyll, with the resulting oxaloacetate being predominantly reduced to malate by an NADP+-specific malate dehydrogenase. The malate is transported to the bundle sheath chloroplasts and decarboxylated to pyruvate and CO, by "malic" enzyme. The CO2 is refixed by RuDP carboxylase and further metabolized through the Calvin cycle (4,7,8,16,21,32). In marked contrast to this compartmentation scheme is that recently proposed by Coombs and co-workers (6, 9, 14, 15) and supported by Laetsch and Kortschak (25). These authors propose an identical reaction sequence for C, photosynthesis, but suggest that all the enzymes for photosynthetic carbon metabolism are compartmented between the mesophyll cytoplasm or nongreen leaf cells and the mesophyll chloroplasts, relegating the bundle sheath to a mere amyloplast-like function. A third scheme of C4 photosynthesis, suggesting that photosynthetic carbon metabolism is similar in both cell types, has also been proposed (29).The purpose of our studies has been to re-examine the role of bundle sheath cells in C4 photosynthesis by using a highly purified preparation of bundle sheath strands isolated from fully expanded leaves of maize. We have previously reported on the effects of oxygen on maize bundle sheath photosynthesis (12,13). A more complete description of the photosynthetic carbon metabolism of these bundle sheath strand preparations is the subject of this report.MATERIALS AND METHODS Plant Materials. Zea mays L., var. W23/L317, seeds were germinated and grown in a soil-sand-peat moss mixture in a growth chamber with a 12-hr day at 25 C and a 12-hr night at 20 C. Light of about 2000 ft-c at the leaf surface was provided by incandescent and cool white fluorescent lamps. Only fully expanded primary and secondary leaves from 11-to 14-day-old seedlings were used. For some of the enzyme assays, crude leaf extracts from the C3 plant soybean (Gb'cine max [L.] Merrill, vars. Kent and Waseda) were also prepared. Leaf material was harvested from the youngest, fully ex...

Differential biogenesis of photosystem‐II in mesophyll and bundle‐sheath cells of ‘malic’ enzyme NADP+‐type C4 plants

“…With the young maize seedlings. the mechanical shearing procedure as originally introduced by Chollet and Ogren [30] was found to be suitable. From the virtual absence of the mesophyll marker enzymes phosphoenolpyruvate carboxylase and malate dehydrogenase (NADP'), bundle-sheath fractions obtained by this procedure were essentially free of mesophyll components (Fig.…”

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C4 Plant Zea mays

“…The ratios are rather higher than reported elsewhere for C4 species ofthe NADP-ME type and this was apparently due to the revised equations (19) used here for Chl estimations. Chl a/b ratios of about 6 compared to about 3 for mesophyll chloroplasts and about 4 for leaf extracts have been reported for bundle sheath cell extracts (6,7,21,24), bundle sheath chloroplasts which were capable of photophosphorylation (2), and chloroplast fragments (1, 1 1). The high ratio for C4 bundle sheath chloroplasts of the NADP-ME type has been attributed to the lack of PSII and its associated…”

“…The evidence for these functions is largely based on enzyme localization studies using chloroplasts isolated from maize by nonaqueous techniques (30), more recently from bundle sheath protoplasts ofFlaveria trinervia (27), and from studies 'Abbreviations: PCR, photosynthetic carbon reduction; PGA, 3-phosphoglyceric acid; PEP, phosphoenolpyruvate; RuBP, ribulose-l1,5-bisphosphate; Ru5P, ribulose-5-phosphate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; R5P, ribose-5-phosphate; ME, malic enzyme. ofthe metabolic activities of isolated bundle sheath cells ofmaize (5,7,12,14) and other C4 species of the NADP-ME type (6, 12, 26, 29). Other than one brief report (29) it has not been possible to confirm these proposed metabolic activities using functional bundle sheath chloroplasts isolated from NADP-ME type C4 species due to the difficulty in preparing these organelles (see Edwards and Huber [8]).…”

“…Since bundle sheath chloroplasts from NADP-ME-type C4 species have diminished PSII, (see above) it was not possible to measure chloroplast intactness by photosynthetic 02 evolution and ferricyanide exclusion as usual for chloroplasts from other sources (see e.g. Edwards et al [9] (7,21,28), and other C4 species of the NADP-ME type (6,12,23,27). High activities of NADP-ME and the PCR cycle enzymes RuBP carboxylase, Ru5P kinase, PGA kinase, and NADP-GAP dehydrogenase were evident in the chloroplasts and these specific activities increased during Percoll enrichment.…”

Growth and Development of Soybean (Glycine max [L.] Merr.) Pods

“…1 . 1 .31) was assayed in a similar man-p,Ci/,urmole), and 2 mm PEP were included in the reaction medium in place of Ru-P2 and 20 mm NaH"4CO,,`4CO2 fixation was terminated by injecting 0.1 ml of a saturated solution of 2, 4-dinitropheny1hydrazine in 6 N HCl, and aliquots were dried under N2 at 25 C. P-glycolate phosphatase (EC 3.1 .3.18) activity in the uncentrifuged extract was assayed by the release of Pi from P-glycolate as described elsewhere (4).…”