Natural variation in photosynthetic capacity, growth, and yield in 64 field-grown wheat genotypes.

Increasing photosynthesis in wheat has been identified as an approach to enhance crop yield, with manipulation of key genes involved in electron transport and the Calvin cycle as one avenue currently being explored. However, natural variation in photosynthetic capacity is a currently unexploited genetic resource for potential crop improvement. Using gas-exchange analysis and protein analysis, the existing natural variation in photosynthetic capacity in a diverse panel of 64 elite wheat cultivars grown in the field was examined relative to growth traits, including biomass and harvest index. Significant variations in photosynthetic capacity, biomass, and yield were observed, although no consistent correlation was found between photosynthetic capacity of the flag leaf and grain yield when all cultivars were compared. The majority of the variation in photosynthesis could be explained by components related to maximum capacity and operational rates of CO2 assimilation, and to CO2 diffusion. Cluster analysis revealed that cultivars may have been bred unintentionally for desirable traits at the expense of photosynthetic capacity. These findings suggest that there is significant underutilized photosynthetic capacity among existing wheat varieties. Our observations are discussed in the context of exploiting existing natural variation in physiological processes for the improvement of photosynthesis in wheat.

Genetic and management approaches to boost UK wheat yields by ameliorating water deficits.

Faced with the challenge of increasing global food production, there is the need to exploit all approaches to increasing crop yields. A major obstacle to boosting yields of wheat (an important staple in many parts of the world) is the availability and efficient use of water, since there is increasing stress on water resources used for agriculture globally, and also in parts of the UK. Improved soil and crop management and the development of new genotypes may increase wheat yields when water is limiting. Technical and scientific issues concerning management options such as irrigation and the use of growth-promoting rhizobacteria are explored, since these may allow the more efficient use of irrigation. Fundamental understanding of how crops sense and respond to multiple abiotic stresses can help improve the effective use of irrigation water. Experiments are needed to test the hypothesis that modifying wheat root system architecture (by increasing root proliferation deep in the soil profile) will allow greater soil water extraction thereby benefiting productivity and yield stability. Furthermore, better knowledge of plant and soil interactions and how below-ground and above-ground processes communicate within the plant can help identify traits and ultimately genes (or alleles) that will define genotypes that yield better under dry conditions. Developing new genotypes will take time and, therefore, these challenges need to be addressed now.

Molecular mechanisms of plasminogen activation: bacterial cofactors provide clues.

Plasminogen activation is a key event in the fibrinolytic system that results in the dissolution of blood clots, and also promotes cell migration and tissue remodelling. The recent structure determinations of microplasmin in complex with the bacterial plasminogen activators staphylokinase and streptokinase have provided novel insights into the molecular mechanisms of plasminogen activation and cofactor function. These bacterial proteins are cofactor molecules that contribute to exosite formation and enhance the substrate presentation to the enzyme. At the same time, they modulate the specificity of plasmin towards substrates and inhibitors, making a 'specificity switch' possible.

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases.

BACKGROUND: Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. RESULTS: The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. CONCLUSIONS: We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

Rapid kinetics of an N-terminal mutant of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase.

The transient changes in absorption of visible light upon addition of ribulose 1,5-bisphosphate to Co2(+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase were used to show altered catalytic properties of a mutant form of the enzyme from Anacystis nidulans. The mutant form of the enzyme had a modified N-terminus and a 10-fold greater Km for ribulose 1,5-bisphosphate than the natural cyanobacterial enzyme.

Structure of the complex of the antistasin-type inhibitor bdellastasin with trypsin and modelling of the bdellastasin-microplasmin system.

The serine proteinase plasmin is, together with tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), involved in the dissolution of blood clots in a fibrin-dependent manner. Moreover, plasmin plays a key role in a variety of other activation cascades such as the activation of metalloproteinases, and has also been implicated in wound healing, pathogen invasion, cancer invasion and metastasis. The leech-derived (Hirudo medicinalis) antistasin-type inhibitor bdellastasin represents a specific inhibitor of trypsin and plasmin and thus offers a unique opportunity to evaluate the concept of plasmin inhibition. The complexes formed between bdellastasin and bovine as well as porcine beta-trypsin have been crystallised in a monoclinic and a tetragonal crystal form, containing six molecules and one molecule per asymmetric unit, respectively. Both structures have been solved and refined to 3.3 A and 2.8 A resolution. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure like the tissue kallikrein inhibitor hirustasin. The interaction between bdellastasin and trypsin is restricted to the C-terminal subdomain of bdellastasin, particularly to its primary binding loop, comprising residues Asp30-Glu38. The reactive site of bdellastasin differs from other antistasin-type inhibitors of trypsin-like proteinases, exhibiting a lysine residue instead of an arginine residue at P1. A model of the bdellastasin-microplasmin complex has been created based on the X-ray structures. Our modelling studies indicate that both trypsin and microplasmin recognise bdellastasin by interactions which are characteristic for canonically binding proteinase inhibitors. On the basis of our three-dimensional structures, and in comparison with the tissue-kallikrein-bound and free hirustasin and the antistasin structures, we postulate that the binding of the inhibitors toward trypsin and plasmin is accompanied by a switch of the primary binding loop segment P5-P3. Moreover, in the factor Xa inhibitor antistasin, the core of the molecule would prevent an equivalent rotation of the P3 residue, making exosite interactions of antistasin with factor Xa imperative. Furthermore, Arg32 of antistasin would clash with Arg175 of plasmin, thus impairing a favourable antistasin-plasmin interaction and explaining its specificity.

Adaptation of photosynthesis in marama bean Tylosema esculentum (Burchell A. Schreiber) to a high temperature, high radiation, drought-prone environment.

Marama bean, Tylosema esculentum, is a tuberous legume native to the Kalahari region of Southern Africa where it grows under high temperatures (typical daily max 37 degrees C during growing season) and radiation (frequently in excess of 2000 micromol m(-2) s(-1)) in sandy soils with low rainfall. These conditions might be expected to select for increased water-use efficiency of photosynthesis. However, marama was found to give similar leaf photosynthetic rates to other C3 plants for a given internal leaf CO2 concentration and Rubisco content. Under conditions of increasing drought, no increase in water-use efficiency of photosynthesis was observed, but stomata closed early and preceded any change in leaf water potential. The possibility of subtle adaptations of photosynthetic characteristics to its natural environment were investigated at the level of Rubisco kinetics. The specificity factor of marama Rubisco was slightly lower than that of wheat, but the apparent Km for CO2 in air (Km') was about 20% lower than that of wheat. This is consistent with better adaptation for efficient photosynthesis at high temperatures in marama compared to wheat, although the net benefit is predicted to be very small (<0.5% at 35 degrees C). The sequence of marama rbcL gene shows 27 deduced amino acid residue differences from that for wheat, and the possibility that one or more of these cause the difference in Rubisco Km' is discussed.

The localisation of 2-carboxy-D-arabinitol 1-phosphate and inhibition of Rubisco in leaves of Phaseolus vulgaris L.

A recent controversial report suggests that the nocturnal inhibitor of Rubisco, 2-carboxy-D-arabinitol 1-phosphate (CAIP), does not bind to Rubisco in vivo and therefore that CA1P has no physiological relevance to photosynthetic regulation. It is now proved that a direct rapid assay can be used to distinguish between Rubisco-bound and free CA1P, as postulated in the controversial report. Application of this direct assay demonstrates that CA1P is bound to Rubisco in vivo in dark-adapted leaves. Furthermore, CA1P is shown to be in the chloroplasts of mesophyll cells. Thus, CA1P does play a physiological role in the regulation of Rubisco.

Efficacy of a growth hormone-releasing peptide mimetic in cardiac ischemia/reperfusion injury.

The cardioprotective efficacy of the pyrazolinone-piperidine dipeptide growth hormone secretagogue (GHS) CP-424,391 was studied in an in vivo rabbit model of ischemia and reperfusion. CP-424,391 was administered at 25 mg/kg p.o. x 7 days. Ischemia was induced by left coronary artery occlusion for 30 min, after which the heart was reperfused for 2 h. At the end of reperfusion, animals were euthanized and the infarct size was determined. The area at risk of infarct was not different between the control (45.8+/-3.7%, n=6) and CP-424,391-treated groups (36.9+/-4.3%, n=11). The infarct size of the control animals was 49.5+/-7.1% and was significantly (P<0.05) lower in the CP-424,391-treated group (infarct size=17.3+/-3.0). There was a trend, albeit not significant, for the left ventricular function to recover to a greater extent in CP-424,391-treated rabbits. Thus, the treatment of rabbits for 7 days with CP-424,391 was cardioprotective against ischemia/reperfusion injury.

Purification and characterization of a milk clotting protease from Mucor bacilliformis.

An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.

Genetic and agronomic approaches to decreasing acrylamide precursors in crop plants.

Progress in developing genetic and agronomic approaches for reducing the levels of the principal precursors of acrylamide, asparagine and sugars in crop plants is reviewed. The factors that affect asparagine and sugar accumulation, particularly in cereal seeds and potato tubers, are described. Asparagine levels appear to be the key parameter in determining acrylamide formation in processed wheat flour and agronomic strategies for reducing asparagine accumulation in wheat grain are reviewed. Sulphur availability has been shown to be particularly important, with sulphur deprivation causing a dramatic increase in grain asparagine levels and acrylamide risk. Nitrogen availability is also a factor, with increasing nitrogen availability causing grain asparagine levels and acrylamide risk to rise. In potato, attention has been focused on sugars, and there has been some success in reducing sugar accumulation in stored potatoes by genetic modification, with a resultant reduction in acrylamide formation. However, the wisdom or otherwise of this dogma is discussed. Other possible genetic targets for manipulation or development as genetic markers in breeding programmes are reviewed. Plant breeders and farmers are encouraged to exploit the varietal differences in acrylamide risk that have already been identified and to develop good agronomic practice to reduce the levels of acrylamide precursors in cereals and potato.

Plant responses to water stress.

This Special Issue comprises a series of papers that develops the theme of plant responses to water stress, encompassing recent developments at the molecular level, through responses of photosynthesis and metabolism, to their application in crop selection and yield. The consideration of water deficits is particularly timely, given the huge developments in this area in the past decade. This issue specifically sets out to place molecular and physiological processes and their agronomic applications in an environmental context.

Kinetic mechanism for the interaction of Hirulog with thrombin.

Hirulog (D-FPRPGGGGDGDFEEIPEEYL) is a bivalent inhibitor of thrombin consisting of a moiety (D-FPRP) that binds to the active-site cleft and a hirudin-like C-terminal region (DGDFEEIPEEYL) that binds to the positively charged surface groove of thrombin known as the anion-binding exosite. The formation of the thrombin-Hirulog complex was studied using steady-state and rapid kinetics at 37 degrees C. The inhibition constant for Hirulog was found to be 1.9 nM. Hirulog was slowly degraded by thrombin with a kcat value of 0.01 s-1. The formation of the complex resulted in an enhancement of 44% in the intrinsic fluorescence of thrombin. The kinetics of the increase in thrombin fluorescence were described by a double-exponential decay. The dependence of the rate constant for the fast phase on the concentration of Hirulog could be described by the Michaelis-Menten equation with Km and kmax values of 0.75 +/- 0.12 microM and 325 +/- 17 s-1. The data were consistent with a mechanism in which the C-terminal region of Hirulog binds to the anion-binding exosite with a dissociation constant of 0.75 microM in the first step, followed by two intramolecular steps with rate constants of about 300 and 30 s-1. A C-terminal fragment of hirudin was found to compete in the first step confirming that this process corresponded to the binding of the hirudin-like C-terminus of Hirulog to the anion-binding exosite.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescents' preference of source to obtain contraceptive information.

PIP: The Adolescent Study, performed as part of the Child Health and Development Studies (CHDS), included an interview in which the adolescents were asked to respond to questions on social and family planning. The adolescents' responses to 1 particular question of this interview regarding the place or person to whom the adolescent would go for information on pregnancy prevention appears to be of particular pertinence in relation to the current issues regarding "Parental Notification Regulations." Recently, the federal government adopted the regulation that agencies receiving funds under Title 10 of the Public Health Service Act must notify a parent or guardian of an unemancipated minor (17 years of age or younger) within 10 days after a prescription contraceptive drug or device has been issued. The regulation has been opposed by major medical associations and has been challenged in court in a lawsuit brought by the Planned Parenthood Federation of America, Inc., resulting in a permanent injunction barring the government from enforcing or implementing the regulation. The government has appealed this decision. The main focus of the Adolescent Study was on blood pressure measurements of adolescents and their mothers. The study also included anthropometric and spirographic measurements, health interviews, and behavioral and attitudinal questionnaires of adolescents and their mothers. 31.4% of the 15 year old girls chose their mother as the person they would be most likely to go to for more information on pregnancy or pregnancy prevention. This proportion was 23.0% for 16 year old girls and 20.5% among the 17 year olds. The choice of father and mother was reduced from 5.6% for 15 year old girls to 1.8% for the 17 year olds. Father exclusively was never mentioned by the girls. For the boys, both parents were preferred by about 10% of the boys and father alone or mother alone, about equally by 7-8% of the boys. The single 1st choice most often mentioned was birth control clinics, increasing for girls from 20% at 15 years to 34% at 17 years. For boys, the percentage was more constant by age at about 26%. A doctor was mentioned as the primary source of information by about 10% of the aodlescent boys and girls. Besides these choices, either another relative or an adult friend or as a friend of the same age was mentioned by about 30% of the adolescents. The data suggest that even if family relationships are favorable, the majority of adolescents intend to seek advice on conception and contraception primarily outside the family.^ieng

Intron-mediated gusA expression in tritordeum and wheat resulting from particle bombardment.

The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns.

The reactions between active and inactive forms of wheat ribulosebisphosphate carboxylase and effectors.

The processes of activation and deactivation of ribulose-1,5-bisphosphate carboxylase purified from wheat have been investigated. Two forms of the enzyme are indistinguishable in terms of ribulose-1,5-bisphosphate carboxylation and oxidation but exhibit different rates of activation. One form is slowly activated in saturating CO2 and Mg2+ at moderate temperatures (t0.5 approximately 120 min at 25 degrees C), the other form rapidly activated (t0.5 approximately 8 s). In the presence of the effectors 6-phosphogluconate or NADPH, significantly lower concentrations of the activating co-factors can achieve full activation of both enzyme species. However, with another effector, fructose 1,6-bisphosphate, for the slowly activating species the mode of action is the same as with 6-phosphogluconate or NADPH, whereas the activation of the rapidly activating species is significantly inhibited. The substrate, ribulose 1,5-bisphosphate, also inhibits this rapid activation process. A mechanism is proposed for the reactions involving activation that accounts for the differential rates of activation and the response to effectors.

Effect of mutations of residue 340 in the large subunit polypeptide of Rubisco from Anacystis nidulans.

Residues 338-342 at the C-terminal end of loop 6 in the large subunit beta/alpha barrel structure of Rubisco influence specificity towards CO2 and O2. In Anacystis nidulans Rubisco, replacement of alanine 340 by tyrosine or histidine increased the specificity factor by 12-13%, accompanied by a 25-33% fall in Vc, the rate of carboxylation, while replacement by asparagine increased the specificity factor by 9% and Vc by 19%. Other mutations did not significantly alter specificity. Alanine 340 does not interact directly with the bisphosphate substrate, thus replacing it with other residues must have indirect effects on the specificity factor and rate of carboxylation.

Is there scope for improving balance between RuBP-regeneration and carboxylation capacities in wheat at elevated CO2?

Carboxylation and RuBP-regeneration capacities, which determine light-saturated photosynthetic rate, were analysed in leaves of spring wheat (Triticum aestivum L. cv. Minaret) grown under different atmospheric CO2 partial pressure (pCa) and N supply regimes. Capacities were estimated from a large number of gas exchange, Rubisco and ATP-synthase content measurements, and from these, the pCa at which the two capacities are equal was derived, to allow direct comparison with growth pCa. Acclimation of the balance between the two capacities to growth at elevated pCa in wheat was only partial and appears to occur mostly in older flag leaves and at low N. However, in contrast to conclusions drawn from previous analyses of these data, there was evidence of a specific effect of growth at 70 Pa pCa, where carboxylation capacity is reduced more than RuBP-regeneration capacity for a given leaf N content. A model was used to estimate the effects of fluctuations in PPFD and temperature in the growth environment on the optimal balance between these capacities. This showed that the observed balance between carboxylation and RuBP-regeneration capacities in young wheat leaves could be consistent with adaptation to the current, or even the preindustrial pCa.

Incorporation of carbon from photosynthetic products into 2-carboxyarabinitol-1-phosphate and 2-carboxyarabinitol.

The synthesis of 2-carboxy-D-arabinitol-1-phosphate (CA1P), the naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, was studied in leaves of the French bean Phaseolus vulgaris, L. Leaves were supplied with air containing 14CO2 in the light then the plants were transferred to normal air in the light or in the dark. Leaf samples were frozen in liquid nitrogen, ground to a powder and extracted with acid. Lipids, pigments and cations were removed from the extract and CA1P and 2-carboxy-D-arabinitol (CA) recovered by anion exchange chromatography. The CA1P was further purified by its specific binding to purified ribulose-1,5-bisphosphate carboxylase/oxygenase. CA and CA1P were identified by chromatographic properties and n.m.r. spectra. When plants were kept for 15 h in darkness after exposure to 14CO2, up to 2.2% and 5.5% of the radioactivity in the extracts was present in CA1P and CA, respectively. The most radioactivity appeared in these compounds when photosynthesis from 14CO2 took place at low photosynthetic photon flux density (PPFD). Under such conditions, radioactivity was detected in CA1P after only 10 min. During subsequent exposure to normal air (12CO2) at low PPFD the amount of radioactivity in CA1P remained almost constant for 6 h; in darkness the rate of incorporation of radioactivity into CA1P reached a maximum after 2 h and the radioactivity was still increasing 6 h later. At low PPFD, the amount of CA1P in the leaves reached a maximum after 2 h. In darkness, the amount of CA1P began to increase rapidly after a lag of almost 1 h, well ahead of the increase in radioactivity in CA1P.