The effect of oxygen on 14CO2 fixation in mesophyll cells isolated from Digitaria sanguinalis (L.) Scop. leaves

Mesophyll cells were isolated from fully-expanded leaves of $\text{Digitaria sanguinalis}$ (L.) Scop. by a combined maceration-filtration technique. In the presence of pyruvate, photosynthetic ¹⁴CO₂ uptake in the isolated cells was not inhibited by atomospheric levels of oxygen. In contrast, superatmospheric levels of oxygen substantially inhibited the light-dependent fixation of ¹⁴CO₂. These oxygen effects are similar to those observed with intact C4 leaves and suggest that the lack of inhibition of C4 photosynthesis by atmospheric levels of oxygen results from the relative oxygen-insensitivity of the phosphopyruvate carboxylase-CO₂ pump in the mesophyll.     

An in vivo analysis of the effect of SO2 fumigation on photosynthesis in Zea mays.

Fumigation of leaves with SO₂ can reduce the capacity for photosynthetic CO₂ uptake even in the absence of visible symptoms of damage. In vitro studies suggest that this invisible injury to intact leaves could be affected by damage to each of the main stages in the photosynthetic process. Reduced stomatal apertures may also reduce photosynthesis following SO₂ fumigation. The responses of CO₂ uptake by leaves to intercellular CO₂ concentration and to absorbed light provide information for quantitative separation of the in vivo contribution of the different stages of photosynthesis to reduction in overall rate. This study uses these techniques to examine the basis of reduction in CO₂ uptake in Zea mays cv. LG11 leaves following short-term fumigation with SO₂. Fumigation with 33 μmol m^–3 SO₂ for 30 min reduced light saturated CO₂ uptake by about one-third. An even greater reduction in light limited CO₂ uptake was observed and with no significant change in light absorptance this was attributed to a reduced quantum yield of photosynthesis. The light saturated CO₂ uptake rate and the stomatal conductance decreased in parallel. However, the relationship of CO₂ uptake to the intercellular CO₂ concentration suggested that the reduced stomatal conductance did not account for the reduced rate of CO₂ uptake following fumigation. Both the initial slope and plateau of this relationship were significantly reduced, suggesting that both carboxylation efficiency and capacity for regeneration of CO₂ acceptor were diminished by SO₂ fumigation. The operating intercellular CO₂ concentration indicated that both processes were co-limiting, before and after fumigation. The time required for induction of photosynthetic CO₂ uptake on illumination was approximately doubled following SO₂ fumigation, showing that fumigation impairs the ability of the photosynthetic apparatus to adapt to fluctuations in light level.     

Isolation of mesophyll protoplasts from mature leaves of soybeans

A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO₃⁻-dependent photosynthesis; greater than 75 micromoles O₂ evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.     

Response to red and blue lights by electrical currents on the surface of intact leaves

Exposure to red and blue lights caused an increase in electrical currents (0.14 μA cm^-2 for red and 0.05 μA cm^-2 for blue, respectively) flowing on the lower surface of leaves fromCommelina communis. However, no changes were measured in currents from isolated epidermal cells. To determine the influence of the mesophyll on such electrical changes, those cells were infiltrated with photosynthesis inhibitors. Both DCCD treated and control leaf discs showed the same level of response to red light. Epidermal strips were also removed to measure the currents above partially exposed mesophyll cells in order to elucidate the relationship between intact leaves and those mesophyll cells. Changes in current were smaller in the latter type. The partially exposed mesophyll cells of a leaf also showed electrical current changes, but smaller than those of the intact leaf. In DCMU-infiltrated leaf discs, the electrical currents of intact leaves were increased to 0.05 μA cm^-2 in response to red light. For sodium azide-infiltrated leaf discs, however, intact leaves showed no response. Likewise, a measure of photosynthetic efficiency, the Fv/Fm ratio, was reduced to that measured in the control, thereby indicating that photosynthetic activity significantly altered the electrical current for intact leaves. Therefore, these results demonstrate that the current observed from the lower side of intact leaves is related to photosynthetic activity in the mesophyll cells.     

Regulation of chloroplast photosynthetic activity by exogenous magnesium

Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl₂, added in the dark, inhibited CO₂-dependent O₂ evolution by Hordeum vulgare L. and Spinacia oleracea L. (C₃ plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O₂ evolution by Digitaria sanguinalis L. (C₄ plant) mesophyll chloroplasts from 80 to 100%. When Mg²⁺ was added in the light, O₂ evolution was reduced only slightly. O₂ evolution in the presence of phosphoglycerate was less sensitive to Mg²⁺ inhibition than was CO₂-dependent O₂ evolution.

Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg²⁺ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg²⁺ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes.

     

Evidence for Cyclic Photophosphorylation during CO(2) Fixation in Intact Chloroplasts: Studies with Antimycin A, Nitrite, and Oxaloacetate

This study examines the effect of antimycin A and nitrite on ¹⁴CO₂ fixation in intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO₂ fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite or oxaloacetate, but not NH₄Cl, in the presence of antimycin A restored photosynthesis. Antimycin A inhibition, and the subsequent restoration of photosynthetic activities by nitrite or oxaloacetate, was observed over a wide range of CO₂ concentration, light intensity, and temperature. High O₂ concentration (up to 240 micromolar) did not appear to influence the extent of the inhibition by antimycin A, nor the subsequent restoration of photosynthetic activity by nitrite or oxaloacetate. Studies of O₂ exchanges during photosynthesis in cells and chloroplasts indicated that 2 micromolar antimycin A stimulated O₂ uptake by about 25% while net O₂ evolution was inhibited by 76%. O₂ uptake in chloroplasts in the presence of 2 micromolar antimycin A was 67% of total O₂ evolution. These results suggest that only a small proportion of the O₂ uptake measured was directly linked to ATP generation. The above evidence indicates that cyclic photophosphorylation is the predominant energy-balancing reaction during photosynthesis in intact chloroplasts. On the other hand, pseudocyclic O₂ uptake appears to play only a minimal role.     

Photosynthetic characteristics of leaves developed at different irradiances and temperatures: an extension of the current hypothesis

Photosynthetic characteristics at high measurement irradiance were analyzed for single leaves of two C₃ and one C₄ species grown under twenty one combinations of irradiance level, irradiance duration, and air temperature in order to test the idea that photosynthetic characteristies developed by leaves in different environments are controlled by the daily amount of photosynthesis. Photosynthetic rates per unit area and mesophyll conductances at 25°C and air levels of CO₂ and O₂, and parameters for two photosynthesis models were used to characterize the photosynthetic properties of the leaves. Leaves with highest values of the photosynthetic parameters for each species were often developed in environments with irradiance levels below saturation for photosynthesis, and with only 12 hours of irradiance per day. Lower air temperature during growth increased the photosynthetic characteristics for a given irradiance regime. Photosynthetic characteristics had higher correlation coefficients with daily photosynthesis of mature leaves divided by 24-hour leaf elongation rates of young leaves, than with daily photosynthesis alone, indicating that photosynthetic characteristics may be related to a balance between photosynthesis and leaf expansion.     

Inhibition of Photosynthesis by Sulfite in Mesophyll Protoplasts Isolated from Vicia faba L. in Relation to Intracellular Sulfite Accumulation

Inhibition of photosynthesis by Na₂SO₃ in mesophyll protoplastsisolated from Vicia faba leaves and uptake of sulfite by theprotoplasts were examined at various pH values of the incubationmedium containing Na₂SO₃. As the pH of the incubation mediumlowered, the rate of photosynthesis in the protoplasts decreasedand the amount of sulfite taken up by the protoplasts increased.Most of sulfite accumulated in the protoplasts was not metabolizedduring the dark incubation, as measured with an ion chromatograph.Photosynthetic O₂ evolution by the chloroplasts isolated fromVicia mesophyll protoplasts was inhibited by exogenously-appliedNa₂SO₃ over pH region examined (7.4–9.0). The sulfiteconcentration required for a half inhibition of photosynthesisby the isolated chloroplasts was similar to the intracellularsulfite level required for that by the protoplasts. These resultsindicate that the intracellular sulfite accumulated in the protoplastsin an unmetabolized state is responsible for the inhibitionof protoplast photosynthesis. (Received January 24, 1985; Accepted May 29, 1985)     

Photosynthetic characteristics of leaves developed at different irradiances and temperatures: an extension of the current hypothesis.

Photosynthetic characteristics at high measurement irradiance were analyzed for single leaves of two C₃ and one C₄ species grown under twenty one combinations of irradiance level, irradiance duration, and air temperature in order to test the idea that photosynthetic characteristics developed by leaves in different environments are controlled by the daily amount of photosynthesis. Photosynthetic rates per unit area and mesophyll conductances at 25°C and air levels of CO₂ and O₂, and parameters for two photosynthesis models were used to characterize the photosynthetic properties of the leaves. Leaves with highest values of the photosynthetic parameters for each species were often developed in environments with irradiance levels below saturation for photosynthesis, and with only 12 hours of iradiance per day. Lower air temperature during growth increased the photosynthetic characteristics for a given irradiance regime. Photosynthetic characteristics had higher correlation coefficients with daily photosynthesis of mature leaves divided by 24-hour leaf elongation rates of young leaves, than with daily photosynthesis alone, indicating that photosynthetic characteristics may be related to a balance between photosynthesis and leaf expansion.     

Impairment of gas exchange and structure in birch leaves (Betula pendula) caused by low ozone concentrations

Summary Injury caused by low O₃ concentrations (0, 0.05, 0.075, 0.1 l 1^-1) was analyzed in the epidermis and mesophyll of fully developed birch leaves by gas exchange experiments and low-temperature SEM: (I) after leaf formation in O₃-free and ozonated air, and (II) after transferring control plants into ozonated air. In control leaves, autumnal senescence also was studied in O₃-free air (III). As O₃ concentration increased, leaves of (I) stayed reduced in size, but showed increased specific weight and stomatal density. The declining photosynthetic capacity, quantum yield and carboxylation efficiency lowered the light saturation of CO₂ uptake and the water-use efficiency (WUE). Carbon gain was less limited by the reduced stomatal conductance than by the declining ability of CO₂ fixation in the mesophyll. The changes in gas exchange were related to the O₃ dose and were mediated by narrowed stomatal pores (overriding the increase in stomatal density) and by progressive collapse of mesophyll cells. The air space in the mesophyll increased, preceded by exudate formation on cell walls. Ozonated leaves, which had developed in O₃-free air (II), displayed a similar but more rapid decline than the leaves from (I). In senescent leaves (III), CO₂ uptake showed a similar decrease as in leaves with O₃ injury but no changes in mesophyll structure and WUE. The nitrogen concentration declined only in senescent leaves in parallel with the rate of CO₂ uptake. A thorough understanding of O₃ injury and natural senescence requires combined structural and functional analyses of leaves.     

Photosynthesis in intact leaves of C3 plants: Physics,physiology and rate limitations

The instantaneous rate of photosynthetic CO₂ assimilation in C₃ plants has generally been studied in model systems such as isolated chloroplasts and algae. From these studies and from theoretical analyses of gas exchange behavior it is now possible to study the biochemistry of photosynthesis in intact leaves using a combination of methods, most of which are nondestructive. The limitations to the rate of photosynthesis can be divided among three general classes: (1) the supply or utilization of CO₂, (2) the supply or utilization of light, and (3) the supply or utilization of phosphate. The first limitation is most readily studied by determining how the CO₂ assimilation rate varies with the partial pressure of CO₂ inside the leaf. The second limitation can be studied by determining the quantum requirement of photosynthesis. The third limitation is most easily detected as a loss of O₂ sensitivity of photosynthesis. Measurement of fluorescence from intact leaves can give additional information about the various limitations. These methods are all non-destructive and so can be observed repeatedly as the environment of a leaf is changed. In addition, leaves can be quick-frozen and metabolite concentrations then measured to give more information about the limitations to intact leaf photosynthesis rates. In this review the physics and biochemistry of photosynthesis in intact C₃ leaves, and the interface between physiology and photosynthesis—triose phosphate utilization—are discussed.     

Photosynthesis in chloroplasts rapidly isolated from primary leaves of oat (Avena sativa L.): effects of orthophosphate, metal ion and chelators

Abstract A simple mechanical method for the rapid isolation of chloroplasts with high rates of photosynthesis from young leaves of oat (Avena sativa L.) was described. The photosynthetic activity of these chloroplasts was stable for at least 2 h with rates of CO₂-dependent O₂ evolution of 30–40 μmol g ¹ Chl s ¹. The photosynthetic properties of these chloroplasts were similar to those reported for spinach and pea chloroplasts isolated by mechanical disruption. The pH optimum for photosynthetic O₂ evolution was pH 7.6. The induction time was 0.5–2 min. Maximal rates of photosynthetic O₂ evolution in these chloroplast preparations were obtained in the absence of both divalent cations and EDTA. Addition of divilent cations strongly inhibited photosynthesis which could be partially restored by the subsequent addition of EDTA. But when these cations were not present in the assay medium the addition of EDTA greater than 1 mol m ³ decreased photosynthetic activity. The optimal orthophosphate concentration required for photosynthesis in these chloroplast preparations was 0.2–0.3 mol m ³. In contrast, the addition of pyrophosphate either in the light or dark inhibited photosynthesis. In a comparative study, chloroplasts were also isolated from oat and wheat (Triticum aestivum L., cultivar Hybrid C306) protoplasts. These chloroplast preparations were found to have properties similar to those determined for oat chloroplasts isolated by the mechanical method reported above.     

Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO₂ reduction and were insensitive to additions of photosynthetic intermediates when CO₂ was available at saturating concentrations. Photosynthetic rates were measured polarographically as O₂ evolution, and the extent of the reduction of substrate was estimated from the amount of O₂ evolved. With CO₂ as substrate, inhibition of photosynthesis by O₂ was dependent on pH. At pH values above 8, rates of O₂ evolution were strongly inhibited by O₂ and only a fraction of the added bicarbonate was reduced before O₂ evolution ceased. The extent of O₂ evolution declined with increasing O₂ concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O₂ levels, but the extent of O₂ evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O₂ evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O₂ concentration. In contrast, there was little effect of O₂ and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O₂. The results are discussed in terms of a balance between photosynthetic O₂ evolution and O₂ consumption by the ribulose diphosphate oxygenase reaction.     

Oxygen inhibits maize bundle sheath photosynthesis

Oxygen inhibits photosynthetic CO₂ fixation in isolated maize bundle sheath strands. This inhibition is rapidly and completely reversible and is relieved by increased levels of CO₂. These observations provide evidence that the relative oxygen-insensitivity of photosynthesis in intact maize leaves results from a CO₂ concentrating mechanism.     

Inhibition of photosynthesis ofPopulus euramericana andHelianthus annuus by SO2, NO2 and O3

Populus euramericana cv. I-214 andHelianthus annuus L. cv. Russian Mammoth were exposed to various concentrations of O₃ SO₂ or NO₂ for 2 h in a cylindrical assimilation chamber. The threshold concentrations of air pollutants for inhibition of net photosynthesis differed between the two species and also between the pollutants tested. Furthermore, the lethal concentrations where the net photosynthetic rates were completely inhibited, also differed between species and between pollutants. For SO₂ and NO₂,P. euramericana was more tolerant photosynthetically thanH. annuus when related to the concentration of pollutants used during the experiment. However, when related to the cumulative uptake rate of each pollutant, the photosynthetic tolerance of the two species was similar. In contrast to the effects of SO₂ or NO₂, the influence of O₃ on net photosynthesis was quite different. The relative rates of net photosynthesis in both species showed the same linear relationship with O₃ concentration. However, the relationship between the relative rate of net photosynthesis and the cumulative uptake rate of O₃ differed between the two species, although it was linear in both cases.     

Phenols,phenoloxidase, and photosynthetic activity of chloroplasts isolated from sugar cane and spinach

Summary Sugar cane chloroplasts isolated in simple media possessed little photochemical activity, but showed rapid O₂ uptake, independent of light. A similar rapid consumption of O₂ was observed with brei prepared from cane leaves. This was not observed in brei of spinach leaves. Authentic polyphenols and cane leaf extracts stimulated the consumption of O₂ by cane preparations and inhibited photosynthesis in chloroplasts isolated from spinach. Chlorogenic acid and caffeic acid were the major o-diphenols in extracts of cane leaves. These compounds inhibited reactions associated with CO₂ fixation by the photosynthetic carbon reduction cycle. Assimilation of CO₂ due to phosphoenol pyruvate carboxylase activity was less sensitive to inhibition by o-diphenols. Mechanisms are discussed whereby o-diphenols may inhibit cane chloroplasts during their isolation.     

Seasonal changes in photosynthetic characteristics of Anemone raddeana,a spring-active geophyte,in the temperate region of Japan

Summary Seasonal changes in the photosynthetic characteristics of intact involucral leaves of Anemone raddeana were investigated under laboratory conditions. Net photosynthesis and constant water vapor pressure deficit showed almost the same seasonal trend. They increased rapidly from mid-April immediately after unfolding of the leaves and reached the maximum in late-April, before the maximum expansion of the leaves. They retained the maximum values until early-May and then decreased toward late-May with a progress of leaf senescence. The calculated values of intercellular CO₂ concentration and relative stomatal limitation of photosynthesis showed no significant change throughout the season. The carboxylation efficiency as assessed by the initial slope of Ci-photosynthesis curve and the net photosynthesis under a high Ci regime varied seasonally in parallel with the change of the light-saturated photosynthesis. The results indicate that the seasonal changes in light-saturated net photosynthesis are not due to a change of stomatal conductance, but to a change in the photosynthetic capacity of mesophyll. Nevertheless, leaf conductance changed concomitantly with photosynthetic capacity, indicating that the seasonal change in stomatal conductance is modulated by the mesophyll photosynthetic capacity such that the intercellular CO₂ concentrations is maintained constant. The shape of light-photosynthesis curve was similar to that of sun-leaf type. The quantum yield also changed simultaneously with the photosynthetic capacity throughout the season.Contribution No. 2965 from the Institute of Low Temperature Science     

An improved method for the simultaneous determination of photosynthetic O2 evolution and CO2 consumption in Rhizophora mucronata leaves

The photosynthetic gas-exchange has been assessed traditionally either as O₂ evolution or CO₂ consumption. In this study, we used a liquid-phase O₂ electrode combined with CO₂ optodes to examine simultaneously photosynthesis in intact leaves of mangrove Rhizophora mucronata. We verified suitable conditions for leaf photosynthetic rates by assessing pH levels and NaHCO₃ concentrations and compared these to the gas-exchange method at various PAR levels. The photosynthetic rate in response to pH exhibited a similar pattern both for O₂ evolution and CO₂ consumption, and higher rates were associated with intermediate pH compared with low and high pH values. The net photosynthetic quotient (PQ) of R. mucronata leaves ranged from 1.04–1.28. The PQ values, which were never lesser than 1, suggested that photorespiration did not occur in R. mucronata leaves under aqueous conditions. The similar maximum photosynthetic rates suggested that all measurements had a high capacity to adjust the photosynthetic apparatus under a light saturation condition. The simultaneous measurements of O₂ evolution and CO₂ consumption using the Clark oxygen electrode polarographic sensor with the CO₂ optode sensor provided a simple, stable, and precise measurement of PQ under aqueous and saturated light conditions.     

Photosynthesis in Flaveria brownii, a C(4)-Like Species: Leaf Anatomy, Characteristics of CO(2) Exchange, Compartmentation of Photosynthetic Enzymes, and Metabolism of CO(2)

Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO₂ compensation points at different O₂ levels, which is typical of C₄ plants, yet it does show about 4% inhibition of net photosynthesis by 21% O₂ at 30°C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C₄ pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C₃ cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial ¹⁴C products in whole leaves and extrapolation of pulse-labeling curves to zero time indicated that about 80% of the CO₂ is fixed into C₄ acids (malate and aspartate), whereas about 20% of the CO₂ directly enters the C₃ cycle. This is consistent with the high activity of enzymes for CO₂ fixation by the C₄ pathway and the substantial activity of enzymes of the C₃ cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C₃ photosynthesis in the mesophyll cells and should be considered a C₄-like species.     

Photorespiratory properties of protoplasts from C3-C4 intermediate species of moricandia

Protoplasts were isolated from leaves of the C₃-C₄ intermediate species, Moricandia arvensis (L.) DC. and Moricandia spinosa Pomel. Analysis by light and transmission electron microscopy indicated that these purified preparations contained both mesophyll protoplasts (MP) and bundle-sheath protoplasts (BSP). Conventional density gradient centrifugation procedures failed to yield separations of pure protoplasts from each cell-type. With these heterogeneous suspensions of MP and BSP, values measured for (i) the percentage inhibition of photosynthetic CO₂ fixation by O₂, (ii) the apparent K_m(CO)2 of photosynthesis, and (iii) dark/light ratios of the rate of ¹⁴CO₂ evolution during decarboxylation of exogenous [1-¹⁴C] glycine were not significantly different from those determined for protoplast preparations from related or representative C₃ plants, including M. foetida, Nicotiana tabacum, and Triticum aestivum. In contrast, previous comparisons with C₃ species, using intact leaf tissue from M. arvensis, have shown a reduced sensitivity of net photosynthesis to inhibition by O₂ [Holaday et al., Plant Sci. Lett., 27 (1982) 181] and an enhanced capacity for the photosynthetic refixation of CO₂ evolved during decarboxylation of exogenous photorespiratory substrates [Holbrook et al., Plant Physiol., 77 (1985) 578]. We conclude that these photosynthetic properties, associated with reduced photorespiration by M. arvensis and M. spinosa, are dependent upon the integrity of the anatomical and ultrastructural arrangement of bundle-sheath and mesophyll cells in these C₃-C₄ intermediate species.