Comparative analysis of biochemical properties of mesophyll and bundle sheath chloroplasts from various subtypes of C4 plants grown at moderate irradiance

The photochemical characteristics of mesophyll and bundle sheath chloroplasts isolated from the leaves of C4 species were investigated in Zea mays (NADP-ME type), Panicum miliaceum (NAD-ME type) and Panicum maximum (PEP-CK type) plants. The aim of this work was to gain information about selected photochemical properties of mesophyll and bundle sheath chloroplasts isolated from C4 plants grown in the same moderate light conditions. Enzymatic as well as mechanical methods were applied for the isolation of bundle sheath chloroplasts. In the case of Z. mays and P. maximum the enzymatic isolation resulted in the loss of some thylakoid polypeptides. It was found that the PSI and PSII activities of mesophyll and bundle sheath chloroplasts of all species studied differed significantly and the differences correlated with the composition of pigment-protein complexes, photophosphorylation efficiency and fluorescence emission characteristic of these chloroplasts. This is the first report showing differences in the photochemical activities between mesophyll chloroplasts of C4 subtypes. Our results also demonstrate that mesophyll and bundle sheath chloroplasts of C4 plants grown in identical light conditions differ significantly with respect to the activity of main thylakoid complexes, suggesting a role of factor(s) other than light in the development of photochemical activity in C4 subtypes.     

河西走廊芦苇的光合碳同化途径对生境条件的适应

 本文以甘肃省河西走廊生长的四个不同生境芦苇为对象,比较研究了它们的叶解剖结构、光合关键酶活力、乙醇酸氧化酶活力和稳定碳同位素组成(δ13C)。结果发现,沼泽芦苇叶中虽具有不典型的花环结构,但维管束鞘细胞中不含叶绿体,RUBPcase活力／PEPcase活力比值为24.4,乙醇酸氧化酶活力为1218Unit mgpro-1·min-1δ13C值为-34‰,这些测值位于C3植物(小麦)的范围内。生长于沙丘上的芦苇叶片具有明显的花环结构,维管束鞘细胞内含异型叶绿体,RUBPcase活力／PEPcase活力比值为0.985, 乙醇酸氧化酶活力为504 Unitmgpro-1·min-1,δ13C值为-20.9‰,这些测值与典型C4植物(玉米)十分相似。盐化草甸芦苇和盐化草一沙丘过渡地带芦苇叶中均具有明显的花环结构,维管束鞘细胞中含大型叶绿体,RUBPcase活力／PEPcase活力比值分别为2.45和1.53,但乙醇酸氧化酶活力分别为1470和2058Unitmgpro-1·min-1,δ13C值分别为35.6和30.6‰,综合盐化草甸芦苇和过渡地带芦苇的上述指标,似介于沼泽芦苇和沙丘芦苇之间。由这些结果可以认为,分布于甘肃省河西走廊的芦苇,在种内发生有由环境因子引起的光合碳代谢途径的适应性改变。     

Centripetal Disposition of Bundle Sheath Chloroplasts During the Leaf Development of Eleusine coracana

Distribution of chloroplasts in bundle sheath cells was examinedby light and electron microscopy during the leaf developmentof finger millet (Eleusine coracana Gaertn.), an NAD malic enzymetype C₄ plant with centripetal arrangement of bundle sheathchloroplasts. Young chloroplasts are almost evenly distributedalong the cell walls in bundle sheath cells of folded immatureleaves. In elongating leaves and above the elongation zone thebundle sheath chloroplasts tend to lie along the radial wallsand the walls adjacent to the vascular bundle. They furthermigrate near to the vascular bundle and finally establish acentripetal arrangement. Mitochondria, microbodies and nucleusmigrate along with the chloroplasts. Etioplasts and other organellesare centripetally located in the bundle sheath cells of etiolatedseedlings grown in the dark. Bundle sheath chloroplast, C₄ plant, chloroplast, chloroplast orientation, Eleusine coracana, finger millet     

Activity, location, and role of asparate aminotransferase and alanine aminotransferase isoenzymes in leaves with C4 pathway photosynthesis

Further evidence has been provided that C₄-pathway species characterized by having low malic enzyme activity contain exceptionally high activities of aspartate and alanine aminotransferases. The total activity of both enzymes is distributed about equally between mesophyll and bundle sheath cells. However, the activity in the two cell types is due to different isoenzymes. In addition to the one quantitatively major isoenzyme associated with each cell type there were at least two additional isozymes of each aminotransferase detectable in the different species examined. Increases in activity of both aminotransferases of ten-fold or more were observed during greening of leaves of dark-grown plants. This increased activity was due specifically to the two quantitatively major isoenzymes associated, respectively, with the mesophyll and bundle sheath cells of green leaves, providing further evidence for their specific role in photosynthesis. Apparently, neither the aspartate nor alanine aminotransferases of mesophyll cells was associated with chloroplasts or other subcellular organelles. However, the major aspartate aminotransferase isoenzyme of bundle sheath cells was associated with mitochondria. These findings are discussed in relation to the probable role of aspartate and alanine aminotransferases in C₄-pathway photosynthesis.     

Functional characterization of phosphoenolpyruvate carboxykinase-type C4 leaf anatomy: immuno-, cytochemical and ultrastructural analyses

BACKGROUND AND AIMS: Species having C4 photosynthesis belonging to the phosphoenolpyruvate carboxykinase (PEP-CK) subtype, which are found only in family Poaceae, have the most complex biochemistry among the three C4 subtypes. In this study, biochemical (western blots and immunolocalization of some key photosynthetic enzymes) and structural analyses were made on several species to further understand the PEP-CK system. This included PEP-CK-type C4 species Urochloa texana (subfamily Panicoideae), Spartina alterniflora and S. anglica (subfamily Chloridoideae), and an NADP-ME-type C4 species, Echinochloa frumentacea, which has substantial levels of PEP-CK. KEY RESULTS: Urochloa texana has typical Kranz anatomy with granal chloroplasts scattered around the cytoplasm in bundle sheath (BS) cells, while the Spartina spp. have BS forming long adaxial extensions above the vascular tissue and with chloroplasts in a strictly centrifugal position. Despite some structural and size differences, in all three PEP-CK species the chloroplasts in mesophyll and BS cells have a similar granal index (% appressed thylakoids). Immunolocalization studies show PEP-CK (which catalyses ATP-dependent decarboxylation) is located in the cytosol, and NAD-ME in the mitochondria, in BS cells, and in the BS extensions of Spartina. In the NADP-ME species E. frumentacea, PEP-CK is also located in the cytosol of BS cells, NAD-ME is very low, and the source of ATP to support PEP-CK is not established. CONCLUSIONS: Representative PEP-CK species from two subfamilies of polyphyletic origin have very similar biochemistry, compartmentation and chloroplast grana structure. Based on the results with PEP-CK species, schemes are presented with mesophyll and BS chloroplasts providing equivalent reductive power which show bioenergetics of carbon assimilation involving C4 cycles (PEP-CK and NAD-ME, the latter functioning to generate ATP to support the PEP-CK reaction), and the consequences of any photorespiration.     

Structure and distribution of chloroplasts and other organelles in leaves with various rates of photosynthesis

The ultrastructure and distribution of chloroplasts, mitochondria, peroxisomes, and other cellular constituents have been examined in cross sections of leaves from plants with either high or low photosynthetic capacity. Photosynthetic capacity of a given plant cannot be correlated with the presence or absence of grana in bundle sheath cell chloroplasts, the presence or absence of starch grains in bundle sheath or mesophyll cell chloroplasts, the chloroplast size in bundle sheath or mesophyll cells, or the location of chloroplasts within bundle sheath cells. We conclude that the number and concentration of chloroplasts, mitochondria, and peroxisomes in bundle sheath cells is the most reliable anatomical criterion presently available for determining the photosynthetic capacity of a given plant.     

A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C₄, Crassulacean acid metabolism (CAM) and C₃ types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C₄ type), kalanchoe (CAM type), french bean, and spinach (C₃ type). In the green leaves of C₄ plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C₃ plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate     

Stable Carbon Isotope Ratio and Activities of PEP Carboxylase and PEP Carboxykinase in Pineapple Leaves

Slight flutuation in carbon isotope values were found in counted from top dounward to the 35th in pineapple, Ananas comosus (L.) Merr., but more negative δ13C value (less heavier 13C) was observed in lower position leaves. The average δ 13C value was –12.94‰ in 11 leaves with maximum range of variation as –2.06‰. Similar single peak curves were found between PEPCase and PEP carboxykinase activities with leaves at various positions. Both enzymes reached the maximum activity in 8—11th leaves, then declined in others at lower positions. PEP carboxykinase activity was 3.4 folds higher than PEPCase activity under the present experimental condition (25—30 ℃). The results indicated that metabolic coordination evisted between dark carboxylation and light decarboxylation. For the obligate CAM plant, pineapple, though the carboxylation and decarxylation activities did occur in old leaves, the CAM level change did much, however.     

Photochemical Characteristics of Mesophyll and Bundle Sheath Chloroplasts from C4 Plants

Mesophyll and bundle sheath chloroplasts were isolated by differential grinding from the leaves of two NADP-ME C₄ plants, Setaria italica Beauv. cv. H-1, Pennisetum typhoides S & H. cv. AKP-2, and a NAD-ME C₄ species Amaranthus paniculatus L. The mesophyll chloroplasts of C₄ plants possessed slightly lower K_m for ADP and Pi than those of bundle sheath chloroplasts. The Hill reaction activities and noncyclic photophosphorylation rates of the bundle sheath chloropiasts from S. italica and P. typhoides were less than one-fifth of those by the mesophyll chloroplasts. But the bundle sheath chloroplasts of A. paniculatus exhibited high rates of Hill reaction, cyclic as well as noncyclic photophosphorylation. The pigment- and eyiochrome composition suggested a relative enrichment of PS 1 in bundle sheath chloroplasts of S. italica and P. typhoides. The chain exists in both mesophyll and bundle sheath chloroplasts. As much as 35–52% of leaf chlorophyll was located in the bundle sheath chloroplasts. The photochemical activities of bundle sheath chloroplasts are significant though a major part of leaf photochemical potential is associated with the mesophyll chloroplasts.     

Significant involvement of PEP-CK in carbon assimilation of C4 eudicots

Background and Aims

C₄ eudicot species are classified into biochemical sub-types of C₄ photosynthesis based on the principal decarboxylating enzyme. Two sub-types are known, NADP-malic enzyme (ME) and NAD-ME; however, evidence for the occurrence or involvement of the third sub-type (phosphoenolpyruvate carboxykinase; PEP-CK) is emerging. In this study, the presence and activity of PEP-CK in C₄ eudicot species of Trianthema and Zaleya (Sesuvioideae, Aizoaceae) is clarified through analysis of key anatomical features and C₄ photosynthetic enzymes.

Methods

Three C₄ species (T. portulacastrum, T. sheilae and Z. pentandra) were examined with light and transmission electron microscopy for leaf structural properties. Activities and immunolocalizations of C₄ enzymes were measured for biochemical characteristics.

Key Results

Leaves of each species possess atriplicoid-type Kranz anatomy, but differ in ultrastructural features. Bundle sheath organelles are centripetal in T. portulacastrum and Z. pentandra, and centrifugal in T. sheilae. Bundle sheath chloroplasts in T. portulacastrum are almost agranal, whereas mesophyll counterparts have grana. Both T. sheilae and Z. pentandra are similar, where bundle sheath chloroplasts contain well-developed grana while mesophyll chloroplasts are grana deficient. Cell wall thickness is significantly greater in T. sheilae than in the other species. Biochemically, T. portulacastrum is NADP-ME, while T. sheilae and Z. pentandra are NAD-ME. Both T. portulacastrum and Z. pentandra exhibit considerable PEP-CK activity, and immunolocalization studies show dense and specific compartmentation of PEP-CK in these species, consistent with high PEP-CK enzyme activity.

Conclusions

Involvement of PEP-CK in C₄ NADP-ME T. portulacastrum and NAD-ME Z. petandra occurs irrespective of biochemical sub-type, or the position of bundle sheath chloroplasts. Ultrastructural traits, including numbers of bundle sheath peroxisomes and mesophyll chloroplasts, and degree of grana development in bundle sheath chloroplasts, coincide more directly with PEP-CK recruitment. Discovery of high PEP-CK activity in C₄ Sesuvioideae species offers a unique opportunity for evaluating PEP-CK expression and suggests the possibility that PEP-CK recruitment may exist elsewhere in C₄ eudicots.     

Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase

Cellular localization of photosynthetic enzymes was investigated by immunogold electron microscopy for leaves of nine C4 grasses (three NADP-malic enzyme (NADP-ME)subtype species, three NAD-malic enzyme (NAD-ME) subtype species, and three phosphoenolpyruvate carboxykinase (PCK) subtype species), two C4 sedges (NADP-ME subtype species) and two C4 dicots (an NADP-ME and an NADP/NAD-ME subtype species). In leaves of all species, immunogold labelling was present for phosphoenolpyruvate carboxylase in the cytosol of the mesophyll cells (MC) and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the bundle sheath cells (BSC). However, considerable specific variation was found in the intercellular patterns of labelling for pyruvate orthophosphate dikinase (PPDK). In the NADP-ME grasses, two NAD-ME grasses, and the dicots, significant labelling for PPDK was present in the both the BSC and the MC chloroplasts. In the other NAD-ME grass, the PCK grasses, and the sedges, labelling for PPDK was present almost exclusively in the chloroplasts of the MC. These patterns were observed in the leaves of both young seedlings and mature plants. These results indicate that the accumulation of PPDK in leaves of C4 plants is not necessarily restricted to the MC, although the chloroplasts of the MC accumulate more than those of the BSC.Key words: C4 plants, immunolocalization, phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, ribulose-1,5-bisphosphate carboxylase/oxygenase.      

High light induced accumulation of two isoforms of the CF1 alpha-subunit in mesophyll and bundle sheath chloroplasts of C4 plants

The effect of light irradiance on the amount of ATP synthase alpha-subunit in mesophyll (M) and bundle sheath (BS) chloroplasts of C(4) species such as maize (Zea mays L., type NADP-ME), millet (Panicum miliaceum, type NAD-ME) and guinea grass (Panicum maximum, type PEP-CK) was investigated in plants grown under high, moderate and low light intensities equal to 800, 350 and 50 micromol photons m(-2) s(-1), respectively. The results demonstrate that alpha-subunit of ATP synthase in both M and BS chloroplasts is altered by light intensity, but differently in the investigated species. Moreover, we identified two isoforms of the CF(1) alpha-subunit, called alpha and alpha. The CF(1) alpha-subunit was the major isoform and was present in all light conditions, whereas alpha was the minor isoform in low light. A strong increase in the level of the alpha-subunit in maize mesophyll and bundle sheath thylakoids was observed after 50 h of high light treatment. The alpha and alpha-subunits from investigated C(4) species displayed apparent molecular masses of 64 and 67 kDa, respectively, on SDS/PAGE. The presence of the alpha-subunit of ATPase was confirmed in isolated CF(1) complex, where it was recognized by antisera to the alpha-subunit. The N-terminal sequence of alpha-subunit is nearly identical to that of alpha. Our results indicate that both isoforms coexist in M and BS chloroplasts during plant growth at all irradiances. We suggest the existence in M and BS chloroplasts of C(4) plants of a mechanism(s) regulating the ATPase composition in response to light irradiance. Accumulation of the alpha isoform may have a protective role under high light stress against over protonation of the thylakoid lumen and photooxidative damage of PSII.     

Use of inhibitors to distinguish between C4 acid decarboxylation mechanisms in bundle sheath cells of C4 plants

Both malate and aspartate were decarboxylated at the 4-carbonposition by isolated bundle sheath strands of C₄ plants butto different extents depending upon the species. In Digitariasanguinalis, an NADP-malic enzyme (NADP-ME) species, 100 µMoxalic acid blocked malate decarboxylation through NADP-ME withoutaffecting aspartate decarboxylation which apparently occursthrough NAD-ME. In several phosphoenolpyruvate carboxykinase(PEP-CK) type C₄ species, 200 µM 3-mercaptopicolinic acid(3-MPA), an inhibitor of PEP-CK, specifically inhibited themalate decarboxylation and partially inhibited aspartate decarboxylation.The aspartate decarboxylation insensitive to 3-MPA may occurthrough NAD-ME. Neither inhibitor prevented C₄ acid decarboxylationin bundle sheath cells of NAD-ME species. The inhibitors thusserved to differentiate between the decarboxylation of C₄ acidsin PEP-CK and NADP-ME type C₄ species through their major decarboxylasefrom that of their less active decarboxylation through NAD-ME. ¹ Present address: Department of Biochemistry and Microbiology,Rutgers University, New Brunswick, NJ 08903, U. S. A. (Received January 28, 1977; )     

Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography

Photosystem II (PS II) activity and the localization of ribulose-l,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) were studied in primary leaves of young maize plants ( Zea mays L. cv. Fronica) by tetra-nitro-blue-tetrazoliumchloride reduction and immunolocalization, respectively. In tissue of 3-day-old plants all chloroplasts were structurally identical. From day 4 they developed into their typical appearance of mesophyll and bundle sheath chloroplasts. First PS II-activity was present in both types of chloroplasts. From day 4 it disappeared in bundle sheath chloroplasts concomitant with the loss of grana. RuBP carboxylase on the other hand was only present in bundle sheath chloroplasts at all stages of development. Thus, the control of the development of the photosystems and the Calvin cycle enzymes seem to differ.     

Photosynthetic Metabolism of Aspartate in Mesophyll and Bundle Sheath Cells Isolated from Digitaria sanguinalis (L.) Scop., a NADP-Malic Enzyme C(4) Plant

Mesophyll cells and bundle sheath strands isolated from leaves of the C(4) plant Digitaria sanguinalis (L.) Scop. are capable of utilizing aspartate as a Hill oxidant. The resulting O(2) evolution upon illumination depends on the presence of 2-oxoglutarate, is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and is stimulated by methylamine. The rate of aspartate-dependent O(2) evolution with mesophyll cells was similar to those with phosphoenolpyruvate + CO(2) or with oxalacetate. Amino-oxyacetate, an inhibitor of aspartate aminotransferase, inhibited the aspartate-dependent O(2) evolution. Aspartate aminotransferase and NADP(+) -malate dehydrogenase are located in the mesophyll chloroplasts. These data suggest that aspartate is converted to oxalacetate via aspartate aminotransferase in the chloroplasts of mesophyll cells and that oxalacetate is subsequently reduced to malate, which is coupled to the photochemical evolution of O(2). This suggestion is further verified by the inhibition of phosphoenolpyruvate-dependent (14)CO(2) fixation by aspartate + 2-oxoglutarate, which presumably acts as oxalacetate and competes with phosphoenolpyruvate + CO(2) for NADPH. dl-Glyceraldehyde inhibited aspartate-dependent O(2) evolution in the bundle sheath strands but not in the mesophyll cells. The data indicate that aspartate may be converted to malate in both mesophyll and bundle sheath cells. In NADP(+) -malic enzyme species, aspartate may exist as a C(4)-dicarboxylic acid reservoir which can contribute to the C(4) cycle through its conversion to malate.     

In situ immunofluorescent localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in mesophyll of C4 dicotyledonous plants

The location of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in the leaf mesophyll of some dicotyledonous C4 plants was confirmed by immunofluorescent labelling. The anti-RuBPCO immune serum was obtained by inoculating a rabbit with commercially obtained RuBPCO. Specificity of these antibodies was tested by immunodiffusion, immunoelectrophoresis, and Western blotting. Fresh hand-cuts of leaves from dicotyledonous C4 plants, Amaranthus caudatus, A. dubius, Gomphrena globosa, and Portulaca oleracea, were incubated with the conjugated anti-RuBPCO immune serum and then with a commercial FITC-anti-rabbit IgG conjugate. Nerium oleander was used a control C3 plant pattern and Zea mays as a C4 plant pattern. The immunofluorescent label was distributed in both mesophyll and bundle sheath in all the C4 plants tested. It is an unequivocal proof that in the C4 dicotyledonous plants the RuBPCO is not only located in the chloroplasts of the bundle sheath cells but also in the chloroplasts of the mesophyll cells. In these plants therefore, the C4 pathway cannot exclusively be viewed as an intercellular level concentration mechanism. In the mesophyll cytoplasm, phosphoenolpyruvate carboxylase traps CO2, while in the mesophyll chloroplasts, RuBPCO operates with atmospheric CO2 and CO2 from the C4 decarboxylation step at an intracellular level, which could mean a significant energetic economy. The CO2 from photorespiration could be saved and reincorporated. Location of RuBPCO in the mesophyll and/or bundle sheath chloroplasts is a matter of inter- and intracellular compartmentation which makes another variation of C4 photosynthetic pathway possible. This revised version was published online in September 2006 with corrections to the Cover Date.     

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

Structural and biochemical characterization of the C₃-C₄ intermediate Brassica gravinae and relatives, with particular reference to cellular distribution of Rubisco

On the basis of its CO(2) compensation concentration, Brassica gravinae Ten. has been reported to be a C(3)-C(4) intermediate. This study investigated the structural and biochemical features of photosynthetic metabolism in B. gravinae. The cellular distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was also examined in B. gravinae, B. napus L. (C(3)), Raphanus sativus L. (C(3)), and Diplotaxis tenuifolia (L.) DC. (C(3)-C(4)) by immunogold electron microscopy to elucidate Rubisco expression during the evolution from C(3) to C(3)-C(4) intermediate plants. The bundle sheath (BS) cells of B. gravinae contained centrifugally located chloroplasts as well as centripetally located chloroplasts and mitochondria. Glycine decarboxylase P-protein was localized in the BS mitochondria. Brassica gravinae had low C(4) enzyme activities and high activities of Rubisco and photorespiratory enzymes, suggesting that it reduces photorespiratory CO(2) loss by the glycine shuttle. In B. gravinae, the labelling density of Rubisco was higher in the mesophyll chloroplasts than in the BS chloroplasts. A similar cellular pattern was found in other Brassicaceae species. These data demonstrate that, during the evolution from C(3) to C(3)-C(4) intermediate plants, the intercellular pattern of Rubisco expression did not change greatly, although the amount of chloroplasts in the BS cells increased. It also appears that intracellular variation in Rubisco distribution may occur within the BS cells of B. gravinae.