Sulfur Assimilation in C₄ Plants

Abstract

The activity of adenosine 5' triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, 35So42-incorporation into adenosine 5' phosphosulfate, and ATP synthesis dependent upon adenosine 5' phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assinilation in relationship to N03-and CO2 assimilation in leaves of C4 plants.Higher plants assimilate the essential elements carbon, nitrogen, and sulfur, primarily as the oxides C02, NO3-, and s042-. The transformations of these oxides into organic molecules are coupled to light-dependent reduction and ATP synthesis reactions in leaves. In plants with the C4 pathway of CO2 assimilation, the transformation of CO2 and NO3 is compartmentalized into specific cell types. While atmospheric CO2 initially is fixed into organic acids in the mesophyll cells of C4 plants, the carboxylation of CO2 to form 3-PGA3 occurs exclusively in the bundle sheath cells (6). Conversely N03-, which is made available to the chlorenchyma cells of plants by way of the transpiration stream, moves past the bundle sheath cells of C4 plants and is reduced exclusively in the mesophyll cells (7,17). The cellular compartmentation of carbon-and nitrogen-assimilating enzymes is a primary factor contributing to high rates of carbon assimilation (6) and nitrogen use efficiency (7,9,17) . In some instances the tissue slices were preincubated in 1% pectinase and 1% cellulysin to increase the yield of cell types. The procedure followed was that described by Chen et al. (11) In a control to correct for nonspecific ATPase activity, NaCl was used in place of Na2MoO4. After incubation for timed periods at 37 C, the reaction was stopped-by the addition of 0.5 ml of ice cold 15% trichloroacetic acid. The reaction tubes were transferred to ice. The tubes were centrifuged at 3,000g for 10 min at 4 C and an aliquot (generally 0.4 ml) was removed for Pi determination.

Initiation of Sulfate Activation: A Variation in C 4 Photosynthesis Plants

“…In all species, more than 95 percent of the activity is associated with the bundle sheath extracts (6). This finding is consistent with our report that in Digitaria sanguinalis ATP sulfurylase was enriched in the bundle sheath cell fraction (7). The whole leaf activities reported in Table 1 are intermediate between the mesophyll and bundle sheath activities, consistent with an enrichment of activity in the bundle sheath, The whole leaf activities also are within the same activity ranges found in C3 and CAM species reported here (Table 1) and previously (5,8).…”

“…It is not known whether the entire process of sulfur assimilation to the reduced level of cysteine is compartmentalized in leaves of C4 plants. Recently, thiosulfonate reductase sulfite reductase activity was shown in both mesophyll and bundle sheath cells of D. sanguinalis, with the bundle sheath extracts containing two to three times more activity than did mesophyll cell extracts (7). While the consequences of cellular compartmentation during sulfur assimilation have yet to be examined fully, it is clear that sulfur activation has been modified in the leaves of C4 plants, with the initial step of sulfate activation occurring primarily in one chloroplast type of the leaf.…”

“…Protein extracts were made in 100 mM tris (pH 8.2), 2 mM ,8mercaptoethanol, 1 mM EDTA, and 1 percent PVP-40 (polyvinylpyrrolidone). Activity of ATP sulfurylase was determined by a bioluminescence assay (7). N.D., not detectable.…”

Sulfate assimilation and glutathione synthesis in C4 plants

“…Given the number of cellular processes spatially distributed in C 4 plants, it was not a great surprise that enzymes of sulfate assimilation were also found to be differentially localised (Gerwick and Black 1979;Gerwick et al 1980;Passera and Ghisi 1982;Schmutz and Brunold 1984). Several groups reported that 75-100% of total leaf ATP sulfurylase activity in maize was confined to BSC (Gerwick Passera and Ghisi 1982;Schmutz and Brunold 1984;Burnell 1984).…”

Reconstruction of Metabolic Pathways, Protein Expression, and Homeostasis Machineries across Maize Bundle Sheath and Mesophyll Chloroplasts: Large-Scale Quantitative Proteomics Using the First Maize Genome Assembly

“…11). Older studies using other assays than proteomics revealed that the activities of ATP sulfurylase (ATPS) and adenosine 5#-phosphosulfate sulfotransferase (APR) in maize were confined to BS cells, while Cys synthase (O-acetyl-Ser thiol lyase [OASTL]) activity is found in both cell types but at a higher level in M cells (Gerwick and Black, 1979;Burnell, 1984;Schmutz and Brunold, 1984;Burgener et al, 1998). These previous observations are consistent with our current observations for ATPS2 and APR3 and -4, which showed BS-M ratios of 3.86, 9.92, and 19.75, respectively, whereas two OASTL isoforms (also named Cys synthase) were 40% to 100% higher in M chloroplasts than in BS chloroplasts.…”