Maize recombinant non-C<Subscript>4</Subscript> NADP-malic enzyme: A novel dimeric malic enzyme with high specific activity

Among the different isoforms of NADP-malic enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C₄-photosynthesis is the most studied. In the present work, the expression in E. coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented. The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the enzyme previously purified from etiolated leaves and roots. Moreover, the recombinant non-photosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a non-C₄ NADP-ME. Using antibodies against this recombinant enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed invivo. The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the enzyme is found in maize roots in vivo. In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2gene and the likely precursor to the evolution of the photosynthetic C₄ NADP-ME) and the 62 kDa isoform (implicated in C₄ photosynthesis). The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C₄ isoenzyme in maize.     

Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.     

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C₄ NADP-malic enzyme species in which the C₄ and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C₄ system. Using differential display, mRNA populations from C₃ and C₄ H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C₄ leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C₄ upregulated genes were identified. Among these one encoded a previously recognized C₄ phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C₄ system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C₄ system in a C₃ crop species.Srinath K. Rao and Hiroshi Fukayama contributed equally to this study.     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Cellular expression of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic enzymes in the amphibious sedge<Emphasis Type="Italic"> Eleocharis retroflexa</Emphasis> ssp.<Emphasis Type="Italic"> chaetaria</Emphasis>

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C₄-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C₃ and C₄ enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C₄ pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C₄ pattern expression in this plant.     

Maize C4 and non-C4 NADP-dependent malic enzymes are encoded by distinct genes derived from a plastid-localized ancestor

NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, an NADP-ME type C4 plant, the most abundant NADP-ME form is the chloroplastic leaf isoform that delivers CO₂ intracellularly to ribulose bisphosphate carboxylase (RuBPCase). A second NADP-ME isoform predominates in maize roots and exhibits distinct C3-like enzymatic characteristics. We show that the C3-like isoform is encoded by a pair of nearly identical genes that encode precursor proteins with functional chloroplast transit peptides. Using RT-PCR, we also show that the messages encoding the C4 and C3-like NADP-ME isoforms are differentially regulated with respect to the developmental stage of the leaf, light conditions, and tissue type. Based on these characteristics and on sequence comparison of ME families in other species, we propose a scheme for the origin of the C4-specific NADP-ME gene.     

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA<Subscript>2</Subscript> (F6a) from <Emphasis Type="Italic">Crotalus durissus collilineatus</Emphasis> Snake Venom

A new crotoxin B isoform PLA₂ (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA₂ (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA₂ (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA₂ (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA₂s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA₂s. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA₂ (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA₂ (F6a) was clearly cytotoxic to these cells.     

Sulfate assimilation and glutathione synthesis in C<Subscript>4</Subscript> plants

Sulfate assimilation and glutathione synthesis were traditionally believed to be differentially compartmentalised in C₄ plants with the synthesis of cysteine and glutathione restricted to bundle sheath and mesophyll cells, respectively. Recent studies, however, showed that although ATP sulfurylase and adenosine 5′ phosphosulfate reductase, the key enzymes of sulfate assimilation, are localised exclusively in bundle sheath in maize and other C₄ monocot species, this is not true for the dicot C₄ species of Flaveria. On the other hand, enzymes of glutathione biosynthesis were demonstrated to be active in both types of maize cells. Therefore, in this review the recent findings on compartmentation of sulfate assimilation and glutathione metabolism in C₄ plants will be summarised and the consequences for our understanding of sulfate metabolism and C₄ photosynthesis will be discussed.     

Differential inhibition of photosynthesis under drought stress in <Emphasis Type="Italic">Flaveria</Emphasis> species with different degrees of development of the C<Subscript>4</Subscript> syndrome

The effect of drought stress (DS) on photosynthesis and photosynthesis-related enzyme activities was investigated in F. pringlei (C₃), F. floridana (C₃–C₄), F. brownii (C₄-like), and F. trinervia (C₄) species. Stomatal closure was observed in all species, probably being the main cause for the decline in photosynthesis in the C₃ species under ambient conditions. In vitro ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and stromal fructose 1,6-bisphosphatase (sFBP) activities were sufficient to interpret the net photosynthetic rates (P _N), but, from the decreases in P _N values under high CO₂ (C _a = 700 μmol mol^{− 1}) it is concluded that a decrease in the in vivo rate of the RuBPCO reaction may be an additional limiting factor under DS in the C₃ species. The observed decline in the photosynthesis capacity of the C₃–C₄ species is suggested to be associated both to in vivo decreases of RuBPCO activity and of the RuBP regeneration rate. The decline of the maximum P _N observed in the C₄-like species under DS was probably attributed to a decrease in maximum RuBPCO activity and/or to decrease of enzyme substrate (RuBP or PEP) regeneration rates. In the C₄ species, the decline of both in vivo photosynthesis and photosynthetic capacity could be due to in vivo inhibition of the phosphoenolpyruvate carboxylase (PEPC) by a twofold increase of the malate concentration observed in mesophyll cell extracts from DS plants.     

C<Subscript>4</Subscript> grasses in boreal fens: their occurrence in relation to microsite characteristics

C₄ plants are rare in cool climates, an ecological pattern attributable to their poor photosynthetic performance at low temperatures relative to C₃ species. However, some C₄ species are able to persist at high latitudes and high elevations, possibly due to the characteristics of the particular microsites they inhabit in these otherwise unfavourable environments. One such species is Muhlenbergia glomerata, which occurs above 60°N in Canada and is found in the atypical C₄ habitat of boreal fens. In this study, we evaluate how microsite features affect the success of M. glomerata in boreal fens. We surveyed 19 populations across northern Ontario during the summers of 1999 and 2000. The ground coverage by woody vegetation was the most important parameter affecting the presence or absence of M. glomerata. Woody plants covered over 50% of the ground area in plots where M. glomerata is absent, but less than 20% where it is present. The minimum light intensity threshold for the presence of the C₄ species was about 32% of full-sunlight at plant height. Surprisingly, in boreal fens M. glomerata was largely restricted to the wetter moss hollows, rather than occurring on the dry hummocks where its greater water use efficiency might have been advantageous. Woody species dominated the hummocks, but were uncommon in the hollows. In these cool northern climates M. glomerata apparently persists because sufficient periods of temperatures favourable to C₄ photosynthesis occur, but this persistence likely requires some factor that suppresses the woody vegetation.     

Enhancing yield of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by controlling NH<Subscript>4</Subscript><Superscript>+</Superscript> concentration

Yield of S-adenosylmethionine was improved significantly in recombinant Pichia pastoris by controlling NH₄ ⁺ concentration. The highest production rate was 0.248 g/L h when NH₄ ⁺ concentration was 450 mmol/L and no repression of cell growth was observed. Within very short induction time (47 h), 11.63 g/L SAM was obtained in a 3.7 L bioreactor.     

Evolution of the C<Subscript>4</Subscript> phosphoenolpyruvate carboxylase promoter of the C<Subscript>4</Subscript> species <Emphasis Type="Italic">Flaveria trinervia</Emphasis>: the role of the proximal promoter region

Background  

The key enzymes of photosynthetic carbon assimilation in C₄ plants have evolved independently several times from C₃ isoforms that were present in the C₃ ancestral species. The C₄ isoform of phosphoenolpyruvate carboxylase (PEPC), the primary CO₂-fixing enzyme of the C₄ cycle, is specifically expressed at high levels in mesophyll cells of the leaves of C₄ species. We are interested in understanding the molecular changes that are responsible for the evolution of this C₄-characteristic PEPC expression pattern, and we are using the genus Flaveria (Asteraceae) as a model system. It is known that cis-regulatory sequences for mesophyll-specific expression of the ppcA1 gene of F. trinervia (C₄) are located within a distal promoter region (DR).     

Deriving room temperature excitation spectra for photosystem I and photosystem II fluorescence in intact leaves from the dependence of <Emphasis Type="Italic">F</Emphasis><Subscript>V</Subscript>/<Emphasis Type="Italic">F</Emphasis><Subscript>M</Subscript> on excitation wavelength

The F ₀ and F _M level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated (PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of F _V/F _M (=(F _M − F ₀)/F _M) have been derived: within each of the three spectra of F _V/F _M, statistically meaningful variations were detected. Also, at distinct wavelength regions, the F _V/F _M differed significantly between leaf types. From spectra of F _V/F _M, excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of F _V/F _M between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the F _V/F _M at these wavelengths. All three leaves exhibited low values of F _V/F _M around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550–650 nm region, the F _V/F _M in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in maize, which is a NADP-ME C₄ species, than in barley, a C₃ species. Finally, low values of F _V/F _M at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra of the F _V/F _M ratio to derive information on spectral absorption properties of PS I and PS II is discussed.     

C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic pathways and life form types for native species from agro-forestry region,Northeastern China

Of the total 570 species, 194 species in 116 genera and 52 families were found with C₃ photosynthesis, 24 species in 17 genera and 6 families with C₄ photosynthesis, and 2 species in 1 genera and 1 family with CAM photosynthesis. 90 % of the total species can be found in Changbai Mountain flora, more a half (69 %) in North China flora, and about 1/3 in Mongolian flora and Xinan flora, respectively. The occurrence of C₄ species was not as common as that in adjacent grasslands and deserts, but relatively more than in the adjacent forests. Of the total 24 C₄ species, 63 % C₄ species (15 of 24) was found in Gramineae. Nine life form types can be found, reflecting the moist climate in the region, especially the occurrence of epiphyte and liana forms. Relatively more geophyte life form plants suggested the winter in the region was much colder than in grasslands. These indicated that both ecological studies and land management decisions must take into account plant photosynthetic pathway and life form patterns, for both of them are closely related to climatic changes and land use.     

Manipulating the Length of the <Emphasis Type="Italic">b</Emphasis> Subunit F<Subscript>1</Subscript> Binding Domain in F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP Synthase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The peripheral stalk of F₁F₀ ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F₀ to the top of the F₁ sector. The stalk serves as the stator necessary for holding F₁ against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F₁. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F₁F₀ complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F₁F₀ ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F₁F₀ complex by a normal b subunit.     

Glucose-6P dehydrogenase in Chlorella sorokiniana (211/8k): an enzyme with unusual characteristics

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH—EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki_NADPH is 103 μM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki_NADPH decreased to 48 μM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.