Biomedical subjects
Publications and source records attributed to G E Edwards.
A 2-year-old male neutered Scottish Terrier presented with dysuria caused by complete urethral obstruction due to a stricture secondary to catheter-induced trauma. The urethral obstruction was resolved by balloon dilation of the stricture under fluoroscopic guidance.
An important adaptation to CO2-limited photosynthesis in cyanobacteria, algae and some plants was development of CO2-concentrating mechanisms (CCM). Evolution of a CCM occurred many times in flowering plants, beginning at least 15-20 million years ago, in response to atmospheric CO2 reduction, climate change, geological trends, and evolutionary diversification of species. In plants, this is achieved through a biochemical inorganic carbon pump called C4 photosynthesis, discovered 35 years ago. C4 photosynthesis is advantageous when limitations on carbon acquisition are imposed by high temperature, drought and saline conditions. It has been thought that a specialized leaf anatomy, composed of two, distinctive photosynthetic cell types (Kranz anatomy), is required for C4 photosynthesis. We provide evidence that C4 photosynthesis can function within a single photosynthetic cell in terrestrial plants. Borszczowia aralocaspica (Chenopodiaceae) has the photosynthetic features of C4 plants, yet lacks Kranz anatomy. This species accomplishes C4 photosynthesis through spatial compartmentation of photosynthetic enzymes, and by separation of two types of chloroplasts and other organelles in distinct positions within the chlorenchyma cell cytoplasm.
Chloroplasts in bundle sheath cells (BSC) of maize perform photosystem I (PSI)-mediated production of ATP. In this study, the participation of ascorbate (Asc) as an electron donor to PSI in light-induced electron transport in isolated maize BSC was demonstrated. It was found that Asc, at physiological concentrations, rapidly reduced photooxidized reaction center chlorophyll of PSI (P700). The rate of Asc donation of electrons to P700+ reached rates of 50-100 microequivalents (mg Chl)(-1) h(-1) at 70-80 mM ascorbate with methyl viologen as an electron acceptor. Electron transport supported by Asc was coupled with membrane energization, as demonstrated by the light-induced formation of a trans-thylakoid electric field measured by the electrochromic shift of carotenoids. The possible physiological function of Asc-dependent electron transport in bundle sheath chloroplasts of maize, as an electron donor for linear electron flow versus sustaining cyclic electron transport, is discussed.
Plant productivity and yield are dependent on source-sink relationships, i.e. the capacity of source leaves to fix CO2 and the capacity of developing sink tissues and organs to assimilate and convert this fixed carbon into dry matter. Studies from our laboratories as well as others have demonstrated that rice productivity and yield are mainly sink-limited during its development because of limited capacity to utilize the initial photosynthetic product (triose phosphate). This limitation in triose phosphate utilization, evident at both the vegetative and reproductive stages of rice development, may be associated with limited capacity for carbohydrate synthesis in rice leaves (which are poor accumulators of starch) or feedback due to limited sink strength of developing seeds. Strategies in improving triose phosphate utilization by enhancing starch production in leaves and developing seeds by the expression of engineered genes for ADP glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, are discussed.
The induction of Crassulacean acid metabolism in M:esembryanthemum crystallinum was investigated in response to foliar application of gibberellic acid (GA). After 5 weeks of treatment, GA-treated plants showed 1.7- to almost a 4-fold increase of phosphoenolpyruvate carboxylase (PEPcase) activity with a concomitant increase in acid metabolism when compared to control plants. Immunoblot analysis indicated an increase in the PEPcase protein similar to that of salt treatment while Rubisco did not show a similar rise. The results indicate that exogenously applied GA accelerates plant developmental expression of PEPcase and Crassulacean acid metabolism in M: crystallinum.
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Critical to defining photosynthesis in C(4) plants is understanding the intercellular and intracellular compartmentation of enzymes between mesophyll and bundle sheath cells in the leaf. This includes enzymes of the C(4) cycle (including three subtypes), the C(3) pathway and photorespiration. The current state of knowledge of this compartmentation is a consequence of the development and application of different techniques over the past three decades. Initial studies led to some alternative hypotheses on the mechanism of C(4) photosynthesis, and some controversy over the compartmentation of enzymes. The development of methods for separating mesophyll and bundle sheath cells provided convincing evidence on intercellular compartmentation of the key components of the C(4) pathway. Studies on the intracellular compartmentation of enzymes between organelles and the cytosol were facilitated by the isolation of mesophyll and bundle sheath protoplasts, which can be fractionated gently while maintaining organelle integrity. Now, the ability to determine localization of photosynthetic enzymes conclusively, through in situ immunolocalization by confocal light microscopy and transmission electron microscopy, is providing further insight into the mechanism of C(4) photosynthesis and its evolution. Currently, immunological, ultrastructural and cytochemical studies are revealing relationships between anatomical arrangements and photosynthetic mechanisms which are probably related to environmental factors associated with evolution of these plants. This includes interesting variations in the C(4) syndrome in leaves and cotyledons of species in the tribe Salsoleae of the family Chenopodiaceae, in relation to evolution and ecology. Thus, analysis of structure-function relationships using modern techniques is a very powerful approach to understanding evolution and regulation of the photosynthetic carbon reduction mechanisms.
Diversity in photosynthetic pathways in the angiosperm family Chenopodiaceae is expressed in both biochemical and anatomical characters. To understand the evolution of photosynthetic diversity, we reconstructed the phylogeny of representative species of tribe Salsoleae of subfamily Salsoloideae, a group that exhibits in microcosm the patterns of photosynthetic variation present in the family as a whole, and examined the distribution of photosynthetic characters on the resulting phylogenetic tree. Phylogenetic relationships were inferred from parsimony analysis of nucleotide sequences of the internal transcribed spacer regions (ITS) of the 18S-26S nuclear ribosomal DNA of 34 species of Salsola and related genera (Halothamnus, Climacoptera, Girgensohnia, Halocharis, and Haloxylon) and representative outgroups from tribes Camphorosmeae (Camphorosma lessingii, Kochia prostrata, and K. scoparia) and Atripliceae (Atriplex spongiosa). A highly resolved strict consensus tree largely agrees with photosynthetic type and anatomy of leaves and cotyledons. The sequence data provide strong support for the origin and evolution of two main lineages of plants in tribe Salsoleae, with NAD-ME and NADP-ME C(4) photosynthesis, respectively. These groups have different C(4) photosynthetic types in leaves and different structural and photosynthetic characteristics in cotyledons. Phylogenetic relationships inferred from ITS sequences generally agree with classifications based on morphological data, but deviations from the existing taxonomy were also observed. The NAD-ME C(4) lineage contains species classified in sections Caroxylon, Malpigipila, Cardiandra, Belanthera, and Coccosalsola, and the NADP-ME lineage comprises species from sections Coccosalsola and Salsola. Reconstruction of photosynthetic characters on the ITS phylogeny indicates separate NAD-ME and NADP-ME lineages and suggests two reversions to C(3) photosynthesis. Reconstruction of geographic distributions suggests Salsoleae originated and diversified in central Asia and subsequently dispersed to Africa, Europe, and Mongolia. Inferred patterns and processes of photosynthetic evolution in Salsoleae should further our understanding of biochemical and anatomical evolution in Chenopodiaceae as a whole.
Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C(4) photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C(3) and C(4) pathways, (14)C labeling of primary photosynthesis products and (13)C/(12)C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C(4) type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C(3) and C(4) types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C(4) type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C(4) type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C(3) type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C(4) photosynthesis in the family Chenopodiaceae.
A computer model comprising light reactions in PS II and PS I, electron-proton transport reactions in mesophyll and bundle sheath chloroplasts, all enzymatic reactions and most of the known regulatory functions of NADP-ME type C(4) photosynthesis has been developed as a system of differential budget equations for intermediate compounds. Rate-equations were designed on principles of multisubstrate-multiproduct enzyme kinetics. Some of the 275 constants needed (DeltaG(0)' and K (m) values) were available from literature and others (V (m)) were estimated from reported rates and pool sizes. The model provided good simulations for rates of photosynthesis and pool sizes of intermediates under varying light, CO(2) and O(2). A basic novelty of the model is coupling of NADPH production via NADP-ME with ATP production and regulation of the C(3) cycle in bundle sheath chloroplasts. The functional range of the ATP/NADPH ratio in bundle sheath chloroplasts extends from 1.5 to 2.1, being energetically most efficient around 2. In the presence of such stoichiometry, the CO(2) concentrating function can be explained on the basis of two processes: (a) extra ATP consumption for starch and protein synthesis in bundle sheath leads to a faster NADPH and CO(2) import compared with CO(2) fixation in bundle sheath, and (b) the residual photorespiratory activity consumes RuBP by oxygenation, NADPH and ATP and causes the imported CO(2) to accumulate in bundle sheath cells. As a wider application, the model may be used for predicting results of genetic engineering of plants.
The effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme have been examined in different cultivars of Phaseolous vulgaris L. grown under 1 and 12 mM nitrogen. Low nitrogen nutrition reduces chlorophyll and soluble protein contents in the leaves and thus the photosynthesis rate and dry-matter accumulation. Chlorophyll, soluble protein and Rubisco contents and photosynthesis rate are not significantly altered by ambient levels of UV-B radiation (17 microW m-2, 290-320 nm, 4 h/day for one week). Comparative studies show that under high nitrogen, UV-B radiation slightly enhances leaf expansion and dry-matter accumulation in cultivar Pinto, but inhibits these parameters in Vilmorin. These results suggest that the UV-B effect on growth is mediated through leaf expansion, which is particularly sensitive to UV-B, and that Pinto is more tolerant than Vilmorin. The effect of UV-B radiation on UV-B-absorbing compounds and on NADP-malic enzyme (NADP-ME) activity is also examined. Both UV-B radiation and low-nitrogen nutrition enhance the content of UV-B-absorbing compounds, and among the three cultivars used, Pinto exhibits the highest increases and Arroz the lowest. The same trend is observed for the specific activity and content of NADP-ME. On a leaf-area basis, the amount of UV-B-absorbing compounds is highly correlated with the enzyme activity (r2 = 0.83), suggesting that NADP-ME plays a key role in biosynthesis of these compounds. Furthermore, the higher sensitivity of Vilmorin than Pinto to UV-B radiation appears to be related to the activity of NADP-ME and the capacity of the plants to accumulate UV-B-absorbing compounds.
Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO2 assimilation, true rates of photosynthetic O2 evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO2 partial pressure. The extent of stimulation of CO2 assimilation by increasing CO2 or by reducing O2 partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO2, the rates of CO2 assimilation and O2 evolution and the percentage inhibition of photosynthesis by low O2 were higher for the wild type than for the mutants. The relative rates of 14CO2 incorporation into starch under high light and high CO2 followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO2 assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.
The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.
The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or beta-glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobhacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5' deletion analyses of the rpL34 promoter indicated that a 50 bp region located between -179 and -129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3' deletion analyses. A 3' deletion up to -28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to - 104. eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.
A concanavalin A (Con A)-binding polypeptide with a molecular mass of 24 kD (termed "SRgp24") was associated with the intercellular space of Mesembryanthemum crystallinum L. callus. When callus was grown in medium containing between 0 and 100 mM NaCl, SRgp24 was detected by Con A binding. Increasing the NaCl concentration to 200 mM caused a reduction in the amount of SRgp24 within 3 d, and returning the callus to medium without salt resulted in an accumulation of SRgp24. Immunoblot analysis showed that appreciable amounts of SRgp24 accumulated in the leaves when plants were grown under sodium-limiting conditions. Unlike most of the cell-wall Con A-binding proteins in M. crystallinum callus, the carbohydrate moiety of SRgp24 was resistant to endoglycosidase H digestion. After purification of SRgp24, the N terminus was sequenced and found to share 55 to 60% identity with the N terminus of osmotin, a group 5 pathogenesis-related protein (PR-5) that accumulates in salt-adapted tobacco cell suspension. Immunocytochemical assays, with affinity-purified antibodies to SRgp24, indicated that SRgp24 preferentially accumulated in the cell-wall region. We conclude that SRgp24 is a salt-responsive glycoprotein related to the PR-5 family in M. crystallinum.
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NADP-malic enzyme functions in plant metabolism as a decarboxylase of malate in the chloroplast or cytosol. It serves as a source of CO2 for photosynthesis in the bundle sheath chloroplasts of C4 plants and in the cytosol of Crassulacean acid metabolism plants, and as a source of NADPH and pyruvate in the cytosol of various tissues. Mg2+ or Mn2+ is required as a cofactor. The enzyme has a high specificity and low Km for NADP+. It exists as a tetramer which may undergo changes in oligomerization and exhibit hysteresis. Its kinetic properties vary depending on the compartmentation and function of the enzyme. The chloroplast form in C4 plants has a high pH optimum (pH 8) under high malate, which favours the tetramer, whereas lower pH (pH 7) favours the dimer form. Generally, other forms of the enzyme, which are thought to be cytosolic, have lower pH optima than the chloroplast enzyme. In a number of cases these forms have been shown to have allosteric properties with malate as a substrate. Chemical modifications of the plant enzyme suggest sulphydryl, histidine and arginine residues are required for catalysis. Primary sequence studies on the chloroplastic enzyme from C4 plants show significant similarities to cytosolic NADP-ME in plants and animals, including a sequence motif which is indicative of the NADP+ binding site. The possible origin of the chloroplast form of the enzyme is discussed.
The expression of maize (Zea mays) phophoenolpyruvate carboxylase (PPC) gene constructions was studied in transgenic tobacco plants (Nicotiana tabacum). Where transcription was under the control of a maize PPC gene promoter, a low level of aberrantly large PPC transcript was detected. Analysis of this PPC transcript indicated that transcription initiation occurs upstream of the normal site. Despite the aberrant transcription initiation, expression of the PPC transcript was still light-regulated. Higher levels of maize PPC transcript of the correct size were obtained with a chimeric gene construction containing a tobacco (Nicotiana plumbaginifolia) chlorophyll a/b binding protein gene promoter. The PPC activities in the leaves of these transgenic plants were up to twofold higher than those of nontransformed plants. Two forms of PPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form with a high apparent K(m) for phosphoenolpyruvate (maize isozyme), and a second form exhibiting a low apparent K(m) (tobacco isozyme). Biochemical analyses of these plants indicated that the transgenic plants had significantly elevated levels of titratable acidity and malic acid. These biochemical differences did not produce any significant physiological changes with respect to photosynthetic rate or CO(2) compensation point.