Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: Photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

Because in the phloem sap of maize (Zea mays L.) leaves a quarter of the total amino nitrogen can be found as alanine, the capacity of a de novo synthesis of alanine from 3-phosphoglycerate (3-PGA) was studied with isolated bundle sheath (BS) strands of maize. Inasmuch as these cells have retained their plasmodesmatic openings, it was possible to study the formation of alanine from 3-PGA when glutamate and ADP were being added. Alanine synthesis required the existence of the intact cell structure. From the formation of the intermediates, partially released to the medium, the activities of the enzymes of the reaction chain from 3-PGA to alanine could be measured in the intact cells. The results show that in the BS cells the rate of alanine production from pyruvate (0.5 micromole/minute per milligram BS chlorophyll) is more than sufficient to produce one-fourth of the assimilated nitrogen as alanine. be expected to take place in the BS cells, because 3-PGA is formed there by photosynthesis. Moreover, previous studies on photosynthetic induction in leaves of Zea mays (8,24) indicated that in the BS cells the glycolytic pathway from 3-PGA to pyruvate is operating and glutamate is found in BS cells at high concentrations (19). In the present report we investigated the capacity of BS cells to produce alanine from 3-PGA. MATERIALS AND METHODS Plant GrowthZea mays was grown in soil under natural illumination supplemented with incandescent and cool white fluorescent lamps to provide 400 to 700 ,uE m-2 s-' in a glasshouse at 25 to 30/15 to 1 8°C day/night temperature. Preparation of BS StrandsBS strands were isolated from fully expanded leaves of 3 to 4 week old plants by blending 2 mm deribbed leaf segments in a polytron giving 1 s (setting at 7) and 15 s (setting at 5-6) bursts. The blending medium contained 0.3 M sorbitol, 20 mM Hepes-KOH (pH 7.6), 4 mM MgCl2, 2 mM Pi, and 10 mM isoascorbic acid. The crude homogenate was filtered through 1.3 and 1 mm sieves and 600 jim and 280 jim nylon nets. The BS strands were collected by filtration on an 88 jim nylon mesh, washed, resuspended on 100 mL of resuspending medium (0.3 M sorbitol, 20 mM

mentioning

confidence: 99%

“…As a result of this, BS2 strands can be obtained consisting of a segment of vascular bundle surrounded by BS cells of high functional integrity and metabolic competence (3,4). In these cells the plasmodesmata that originally connected the BS cytosol with mesophyll cells (21) are retained in their entirety (2,25,26).…”

mentioning

confidence: 99%

Alanine Synthesis by Bundle Sheath Cells of Maize

Valle

Heldt²

1991

“…The basic procedure for preparing bundle sheath cell strands was very similar to that described previously (2). Except where otherwise specified, 15 g of deribbed leaf tissue was thinly sliced with a razor blade and then blended in a Sorvall Omnimixer in 100 ml of an icecold solution containing 0.3 M sorbitol, 50 mM Hepes-KOH (pH 7.5), 5 mM MgCl2, 2 mm Pi, 5 mm dithiothreitol, 0.5% (w/v) BSA, and 0.2% (w/v) sodium isoascorbate.…”

mentioning

confidence: 99%

Low Bundle Sheath Carbonic Anhydrase Is Apparently Essential for Effective C₄ Pathway Operation

Burnell¹,

Hatch²

1988

102

The concentrating of inorganic carbon in bundle sheath cells is the key function of the unique reactions of the C4 pathway (13). The prime purpose of this process is to favor the carboxylation reaction catalyzed by Rubiscol at the expense of oxygenase activity, thereby eliminating photorespiration. Since CO2 as such is the substrate for Rubisco (5), and since it is the ratio of CO2 to 02 that determines the relative rates of carboxylase and oxygenase activities (1), it is the concentration of the CO2 component of the inorganic carbon which is critical. Recently, we reported that CO2 is the product of each of the three decarboxylase reactions operating in C4 plants (15). Based on the assumption that CO2 is both the product of decarboxylation in bundle sheath cells and the substrate for Rubisco a model was developed which predicted that the inorganic carbon pool generated during steady state photosynthesis would exist predominantly as CO2 (8). That is, a steady state situation could exist where the relative concentrations of CO2 and HCO3-remain far removed from thermodynamic equilibrium.In this model another key assumption for such a steady state where CO2 is the predominant inorganic carbon species was that bundle sheath cells contain little or no carbonic anhydrase. In fact, there is some evidence that the carbonic anhydrase activity in leaves of C4 plants is at least largely associated with mesophyll cells (9,16). Carbonic anhydrase would be essential in mesophyll cells to sustain rapid conversion of CO2 entering the cell to bicarbonate, the substrate for PEP carboxylase (15). However, I Abbreviations: Rubisco, ribulose 1,5-bisphosphate carboxylase-oxygenase; PEP, phosphoenolpyruvate.

“…It is difficult to conceive how such measurements might be obtained with intact leaves but, as recently pointed out (3), isolated bundle sheath cells may provide an alternative system. Rapid flux of C4 acids or C3 compounds into isolated bundle sheath cell strands, as judged by resulting rates of metabolism or photosynthesis, have been observed in several separate studies (4,6,19,26).…”

Section: Abstracimentioning

confidence: 99%

“…Rapid flux of C4 acids or C3 compounds into isolated bundle sheath cell strands, as judged by resulting rates of metabolism or photosynthesis, have been observed in several separate studies (4,6,19,26). Responses to the addition of various other metabolites and inhibitors have also been observed, including compounds as large as adenylates and NAD (3,4,9).During the present study we examined the diffusion of various metabolites into isolated bundle sheath cell strands from C4 species and a procedure was developed for measuring diffusion constants for this process. This diffusive flux apparently occurred via plasmodesmata.…”

mentioning

confidence: 99%

Metabolite Diffusion into Bundle Sheath Cells from C₄ Plants

Weiner¹,

Burnell²,

Woodrow³

et al. 1988

ABSTRACI The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C4 plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabo-lites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between I and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function. (23, 27). This derived value for mesophyll-bundle sheath cell resistance has also been applied to different models and given estimated diffusive losses of stored inorganic carbon from bundle sheath cells of the order of 10% of the CO2 originally generated by C4 acid decarboxylation (14, 21). Clearly, a more precise analysis of C4 pathway function would require more reliable direct measurements for the diffusion characteristics of the mesophyll-bundle sheath cell interface. It is difficult to conceive how such measurements might be obtained with intact leaves but, as recently pointed out (3), isolated bundle sheath cells may provide an alternative system. Rapid flux of C4 acids or C3 compounds into isolated bundle sheath cell strands, as judged by resulting rates of metabolism or photosynthesis, have been observed in several separate studies (4, 6, 19, 26). Responses to the addition of various other metabolites and inhibitors have also been observed, including compounds as large as adenylates and NAD (3, 4, 9). During the present study we examined the diffusion of various metabolites into isolated bundle sheath cell strands from C4 species and a procedure was developed for measuring diffusion constants for this process. This diffusive flux apparently occurred via plasmodesmata. The results are considered in relation to the function of C4 photosynthesis. A key feature of C4 plants is the differential partitioning of photosynthetic reactions between mesophyll and bundle sheath cells (21). As a consequence, there is a need for rapid intercellular transport of certain metabolites between these cells. The prime function of C4 photosynthesis is to concentrate CO2 in bundle sheath cells (1, ...