Comparing simple root phenotyping methods on a core set of rice genotypes.

Interest in belowground plant growth is increasing, especially in relation to arguments that shallow-rooted cultivars are efficient at exploiting soil phosphorus while deep-rooted ones will access water at depth. However, methods for assessing roots in large numbers of plants are diverse and direct comparisons of methods are rare. Three methods for measuring root growth traits were evaluated for utility in discriminating rice cultivars: soil-filled rhizotrons, hydroponics and soil-filled pots whose bottom was sealed with a non-woven fabric (a potential method for assessing root penetration ability). A set of 38 rice genotypes including the OryzaSNP set of 20 cultivars, additional parents of mapping populations and products of marker-assisted selection for root QTLs were assessed. A novel method of image analysis for assessing rooting angles from rhizotron photographs was employed. The non-woven fabric was the easiest yet least discriminatory method, while the rhizotron was highly discriminatory and allowed the most traits to be measured but required more than three times the labour of the other methods. The hydroponics was both easy and discriminatory, allowed temporal measurements, but is most likely to suffer from artefacts. Image analysis of rhizotrons compared favourably to manual methods for discriminating between cultivars. Previous observations that cultivars from the indica subpopulation have shallower rooting angles than aus or japonica cultivars were confirmed in the rhizotrons, and indica and temperate japonicas had lower maximum root lengths in rhizotrons and hydroponics. It is concluded that rhizotrons are the preferred method for root screening, particularly since root angles can be assessed.

QTLs associated with root traits increase yield in upland rice when transferred through marker-assisted selection.

Altering root morphology of rice (Oryza sativa L.) cultivars could improve yields in drought-prone upland ecosystems. Marker-assisted backcross breeding was used to introgress four QTLs for root traits into an upland rice cultivar. The QTLs had previously been identified under experimental conditions in a different genetic background. The introgressed lines and the recurrent parent were grown for 6 years by resource-poor farmers in upland sites in Eastern India and yields recorded. In combination the QTLs significantly increased yield by 1 t ha(-1) under relatively favourable field conditions. In less favourable trials, the QTL effects were not detected due to greater heterogeneity in soil-water availability in very low yielding environments and consequent yield variability. Root studies under controlled conditions showed that lines with the introgressions had longer roots throughout tillering than the recurrent parent (14 cm longer 2 weeks after sowing). Therefore, both improved roots and increased yield can be attributed to the introgression of QTLs. This is the first demonstration that marker-assisted backcross breeding (MABC) to introgress multiple root QTLs identified under controlled conditions is an effective strategy to improve farmers' yields of upland rice. The strategy was used to breed a novel upland rice cultivar that has been released in India as Birsa Vikas Dhan 111.

Assessing the importance of genotype x environment interaction for root traits in rice using a mapping population II: conventional QTL analysis.

Modifying plant root systems is considered a means of crop improvement targeted to low-resource environments, particularly low nutrient and drought-prone agriculture. The identification of quantitative trait loci (QTLs) for root traits has stimulated marker-assisted breeding to this end, but different QTLs have been detected in different populations of the same species, and importantly, in the same population when grown in different experimental environments. The presence of QTL x environment interaction is implicated, and this must be characterised if the utility of the target QTLs is to be realised. Previous attempts to do this suffer from a lack of control over replicate environments and inadequate statistical rigour. The Bala x Azucena mapping population was grown in two replicate experiments of four treatment environments, a control, a low light, a low soil nitrogen and a low soil water treatment. After a 4 weeks growth, maximum root length, maximum root thickness, root mass below 50 cm, total plant dry mass, % root mass and shoot length were measured. A summary of the overall results is presented in an accompanying paper. Here, QTL analysis by composite interval mapping is presented. A total of 145 QTLs were detected, mapping to 37 discrete loci on all chromosomes. Superficial evidence of QTL x E (great difference in LOD score) was tested by single-marker analysis which confirmed QTL x E for five loci representing only five individual trait-loci interactions. Some loci appeared to be stable across environments. Some QTLs were clearly more or less active under low light, low nitrogen or drought. A few notable loci on chromosomes 1, 2, 3, 5, 7 and 9 are briefly discussed. Also discussed are some remaining statistical shortcomings that will be addressed in another companion paper.

Assessing the importance of genotype x environment interaction for root traits in rice using a mapping population. I: a soil-filled box screen.

Altering root system architecture is considered a method of improving crop water and soil nutrient capture. The analysis of quantitative trait loci (QTLs) for root traits has revealed inconsistency in the same population evaluated in different environments. It must be clarified if this is due to genotype x environment interaction or considerations of statistics if the value of QTLs for marker-assisted breeding is to be estimated. A modified split-plot design was used where a main plot corresponded to a separate experiment. The main plot factor had four treatments (environments), which were completely randomized among eight trials, so that each treatment was replicated twice. The sub-plot factor consisted of 168 recombinant inbreed lines of the Bala x Azucena rice mapping population, randomly allocated to the seven soil-filled boxes. The aim of the trial was to quantify QTL x environment interaction. The treatments were chosen to alter partitioning to roots; consisting of a control treatment (high-soil nitrogen, high light and high-water content) and further treatments where light, soil nitrogen or soil water was reduced singly. After 4 weeks growth, maximum root length (MRL), maximum root thickness, root mass below 50 cm, total plant dry mass (%), root mass and shoot length were measured. The treatments affected plant growth as predicted; low nitrogen and drought increased relative root partitioning, low-light decreased it. The parental varieties Bala and Azucena differed significantly for all traits. Broad-sense heritability of most traits was high (57-86%). Variation due to treatment was the most important influence on the variance, while genotype was next. Genotype x environment interaction was detected for all traits except MRL, although the proportion of variation due to this interaction was generally small. It is concluded that genotype x environment interaction is present but less important than genotypic variation. A companion paper presents QTL x environment analysis of data.

Marker-assisted selection to introgress rice QTLs controlling root traits into an Indian upland rice variety.

A marker-assisted back-crossing (MABC) breeding programme was conducted to improve the root morphological traits, and thereby drought tolerance, of the Indian upland rice variety, Kalinga III. This variety, the recurrent parent in the MABC, had not previously been used for quantitative trait locus (QTL) mapping. The donor parent was Azucena, an upland japonica variety from Philippines. Five segments on different chromosomes were targeted for introgression; four segments carried QTLs for improved root morphological traits (root length and thickness) and the fifth carried a recessive QTL for aroma. Some selection was made at non-target regions for recurrent parent alleles. We describe the selection made in three backcross (BC) generations and two further crosses between BC3 lines to pyramid (stack) all five target segments. Pyramids with four root QTLs were obtained in eight generations, completed in 6 years using 3,000 marker assays in a total of 323 lines. Twenty-two near-isogenic lines (NILs) were evaluated for root traits in five field experiments in Bangalore, India. The target segment on chromosome 9 (RM242-RM201) significantly increased root length under both irrigated and drought stress treatments, confirming that this root length QTL from Azucena functions in a novel genetic background. No significant effects on root length were found at the other four targets. Azucena alleles at the locus RM248 (below the target root QTL on chromosome 7) delayed flowering. Selection for the recurrent parent allele at this locus produced early-flowering NILs that were suited for upland environments in eastern India.

Variation in arsenic speciation and concentration in paddy rice related to dietary exposure.

Ingestion of drinking water is not the only elevated source of arsenic to the diet in the Bengal Delta. Even at background levels, the arsenic in rice contributes considerably to arsenic ingestion in subsistence rice diets. We set out to survey As speciation in different rice varieties from different parts of the globe to understand the contribution of rice to arsenic exposure. Pot experiments were utilized to ascertain whether growing rice on As contaminated soil affected speciation and whether genetic variation accounted for uptake and speciation. USA long grain rice had the highest mean arsenic level in the grain at 0.26 microg As g(-1) (n = 7), and the highest grain arsenic value of the survey at 0.40 microg As g(-1). The mean arsenic level of Bangladeshi rice was 0.13 microg As g(-1) (n = 15). The main As species detected in the rice extract were AsIII, DMAV, and AsV. In European, Bangladeshi, and Indian rice 64 +/- 1% (n = 7), 80 +/- 3% (n = 11), and 81 +/- 4% (n = 15), respectively, of the recovered arsenic was found to be inorganic. In contrast, DMAV was the predominant species in rice from the USA, with only 42 +/- 5% (n = 12) of the arsenic being inorganic. Pot experiments show that the proportions of DMAV in the grain are significantly dependent on rice cultivar (p = 0.026) and that plant nutrient status is effected by arsenic exposure.

Yield response to water deficit in an upland rice mapping population: associations among traits and genetic markers.

A population of recombinant inbred rice lines from a cross between the upland japonica cultivar Azucena and the upland indica cultivar Bala was evaluated in a series of upland field experiments. Water stress was imposed during the reproductive stage by managed irrigation during the dry season, while control treatments were maintained in aerobic, well-irrigated conditions. Water deficit resulted in a yield reduction of 17 to 50%. The genetic correlation between stress and control yields was quite high when stress was mild, and the heritability of yield was similar in stress and control treatments across both years of this study. Genetic correlations between secondary traits such as leaf rolling and drying and yield under stress varied from high (leaf drying) to insignificant (leaf rolling). Lines with superior yield tended to have fewer panicles and larger grain size than the high-yielding parent, Bala, even though the panicle number was positively correlated with yield and the thousand-grain weight was not associated with yield for the population as a whole. Analysis of quantitative trait loci (QTLs) for yield and yield components allowed the identification of 31 regions associated with growth or yield components. Superior alleles came from either parent. Several of the regions identified had also been reported for root mass at depth or maximum root length in this population in other studies made under controlled environments, and for leaf drying (LD) in field studies. However, the direction of the effect of QTLs was not consistent, which indicates that there was not necessarily a causal relationship between these secondary traits and performance. We conclude that mapping populations can provide novel insights on the actual relationships between yield components and secondary traits in stress and control environments and can allow identification of significant QTLs for yield components under drought stress.

Oxidative Signals in Tobacco Increase Cytosolic Calcium.

Tobacco (Nicotiana plumbaginifolia) seedlings genetically transformed to express apoaequorin were incubated in h-coelenterazine to reconstitute the calcium-sensitive luminescent protein aequorin. Treatment of these seedlings with hydrogen peroxide resulted in a transient burst of calcium-dependent luminescence lasting several minutes. Even though the hydrogen peroxide stimulus was persistent, the change in cytosolic free calcium concentration ([Ca2+]cyt) was transient, suggesting the presence of a refractory period. When seedlings were pretreated with hydrogen peroxide, there was no increase in [Ca2+]cyt upon a second application, which confirmed the refractory character of the response. Only when the two treatments were separated by 4 to 8 hr was full responsiveness recovered. However, treatment with hydrogen peroxide did not inhibit mobilization of [Ca2+]cyt induced by either cold shock or touching, suggesting that these three signals mobilize different pools of intracellular calcium. To examine whether [Ca2+]cyt is regulated by the redox state of the cytoplasm, we pretreated seedlings with buthionine sulfoximine (to modify cellular glutathione levels) and inhibitors of ascorbate peroxidase. These inhibitors modify the hydrogen peroxide-induced transients in [Ca2+]cyt, which is consistent with their effects on the cellular prooxidant/antioxidant ratio. Treatment with hydrogen peroxide that elicited [Ca2+]cyt increases also brought about a reduction in superoxide dismutase enzyme activity. This reduction could be reversed by treatment with the calcium channel blocker lanthanum. This indicates that there is a role for calcium in plant responses to oxidative stress.

A possible role for calcium in oxidative plant stress.

Disruption of calcium homoeostasis may be crucially important in damaging animal cells under oxidising conditions. In hepatocytes, oxidative stress causes an increase in cytosolic calcium, a process which is thought to be largely responsible for subsequent cell death. The oxidation of glutathione precedes a flux of calcium into the cytoplasm from internal and external sources. The possibility that similar events occur in plant cells has been tested using strips of epidermis taken from leaves of Commelina communis. The closure of stomata in response to many chemical or physical stimuli is thought to involve a transient rise in cytosolic calcium. Paraquat (10(-4) M) and hydrogen peroxide (10(-3) M) caused a marked reduction in stomatal aperture when included in the incubation medium of illuminated epidermal strips. The calcium channel blocker verapamil (10(-5) M) protected stomata from hydrogen peroxide-induced closure while the ion chelator EGTA (2 x 10(-3) M) protected stomata in both oxidising treatments. These results are discussed in so far as they support the hypothesis that a rise in cytosolic calcium may play a role in the pathology of oxidative stress in plant cells through a mechanism analogous to the liver cell system.

Mechanisms of NSAID-induced gastroenteropathy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used and therapeutically effective groups of drugs in the Western world. However, gastrointestinal problems constitute all too frequently reported unwanted effects of NSAIDs; their effects on the gastric (and intestinal) mucosa are, therefore, well documented. A review of the evidence suggests that NSAID-induced gastric damage occurs as a result of a dual insult, by NSAID-mediated direct (and indirect) acidic damage followed almost simultaneously by the deleterious (systemic) effect of prostaglandin inhibition. A number of strategies have been used in the management of NSAID-induced gastric damage; however, not all have been entirely successful. Nonetheless, a number of approaches have met with a considerable amount of success, including direct gastroprotection, enhancement of mucosal defence mechanisms and, perhaps most encouragingly, the development of newer NSAIDs which display not only considerable clinical efficacy but a reduced gastrointestinal adverse event profile.

Salbutamol in the 1980s. A reappraisal of its clinical efficacy.

Salbutamol (albuterol) is a beta 2-selective adrenoceptor agonist which accounts for its pronounced bronchodilatory, cardiac, uterine and metabolic effects. During the intervening years since salbutamol was first reviewed in the Journal (1971), it has become extensively used in the treatment of reversible obstructive airways disease. Numerous studies in this disease (including severe acute, childhood and exercise-induced asthma) have confirmed the bronchodilatory efficacy of salbutamol, and it has been shown to be at least as effective as most of the currently available bronchodilators, if not more effective. The onset of maximum effect of salbutamol is dependent on the formulation used and the route by which it is administered. In most patients inhaled salbutamol is a first-line therapy, since it offers rapid bronchodilation, usually relieving bronchospasm within minutes. Although oral salbutamol has often proved to be less efficacious than the inhaled formulation, it still affords clinically significant bronchodilation, and it is particularly useful in those patients unable to coordinate the use of inhalers. Parenteral formulations of salbutamol are generally reserved for the treatment of severe attacks of bronchospasm and they are one of the treatments of choice in these life-threatening situations. Studies of the concomitant use of salbutamol and other agents such as anticholinergics, methylxanthines and beclomethasone dipropionate have usually shown a complementary response in the majority of patients, as might be expected from the different mechanisms of action of these groups of drugs. Salbutamol is generally well tolerated and any side effects observed are a predictable extension of its pharmacology. Since the frequency of side effects is dose related, and therefore dependent on the route of administration, it is not surprising that they are much more common following intravenous and oral rather than inhalation therapy. Tremor, tachycardia and hypokalaemia are the most frequently reported adverse effects. After nearly 20 years of use, salbutamol is well established as a 'first-choice' treatment in reversible obstructive airways disease. Indeed, throughout this time many new bronchodilatory agents have been studied but none have proved more effective. Clinical evaluation of salbutamol in the treatment of premature labour, hyperkalaemia and cardiac failure awaits further studies, although to date some encouraging results have been reported.

Probucol. A reappraisal of its pharmacological properties and therapeutic use in hypercholesterolaemia.

Probucol is a lipid-regulating agent structurally dissimilar to other known agents, with a unique pharmacodynamic and clinical profile. It is effective in the treatment of primary Type IIa and IIb hyperlipoproteinaemias, including polygenic (non-familial) hypercholesterolaemia and both heterozygous and homozygous forms of familial hypercholesterolaemia, with reductions in plasma total cholesterol and low density lipoprotein (LDL)-cholesterol levels of about 10 to 20% being attained. Marked effects on cutaneous and tendinous xanthomas have been observed, with significant regression often apparent after 2 or 3 months' therapy. Preliminary trials also indicate efficacy in hyperlipoproteinaemia secondary to nephrotic syndrome and diabetes mellitus. The mechanism of the reduction in LDL-cholesterol levels is yet to be fully elucidated, but it is thought that the decrease results from enhanced catabolism, and there is preliminary evidence of an independent antioxidant effect. In contrast with all other known lipid-lowering agents, probucol also effects a consistent reduction in serum high density lipoprotein (HDL)-cholesterol levels, of around 20 to 30%; the clinical significance of this observation is unclear, although some preliminary investigations suggest a beneficial effect in enhancing reverse cholesterol transport. The influence of probucol treatment on cardiovascular morbidity and mortality remains to be fully investigated; a large trial quantifying the potential effect of probucol against the development of atherosclerotic lesions is currently in progress. Adverse effects of probucol are generally mild, seldom requiring treatment withdrawal, with gastrointestinal effects such as diarrhoea predominating. However, indications of an increased frequency of ventricular arrhythmias and sudden death in association with QT interval prolongation in some animals have prompted some concern. Although there is evidence of a degree of QT prolongation in a number of trials in humans, the nature and clinical significance of this effect requires clarification, as no increased incidence of cardiac arrhythmias is apparent. Thus, probucol appears to be of benefit in primary and secondary hyperlipoproteinaemia of Types IIa and IIb, and particularly in homozygous familial hypercholesterolaemia, with marked effects on xanthomas, and a generally favourable adverse effect profile. There is no evidence to date causally relating occasional QT interval prolongation in patients to any incidence of arrhythmias or sudden death. Pharmacodynamic investigations are likely to clarify further the place of probucol in therapy, particularly with respect to its distinctive lowering of plasma HDL-cholesterol levels.

Plants under drought-stress generate activated oxygen.

Lysed chloroplasts prepared from droughted wheat plants generate O2- on illumination as detected by electron spin resonance spectroscopy, the amplitude of the signal increasing with the severity of water deprivation. Following a similar time-course as radical formation and chlorophyll destruction, there was a significant increase in the accumulation of iron in the droughted shoots to reach an estimated concentration in the cell sap of about 2.5 mM. The evidence suggests that superoxide generated as a result of impaired electron transport in the chloroplasts reacts with the high concentration of accumulating iron resulting in the formation of hydroxyl radical, the probable cause of the primary pathologies observed in droughted plants.

Nizatidine. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic use in peptic ulcer disease.

Nizatidine is an H2-receptor antagonist which in animal studies was more active on a weight-for-weight basis than cimetidine in inhibiting basal and stimulated gastric acid secretion. Similarly, studies in humans have confirmed that nizatidine is a potent inhibitor of basal, nocturnal and stimulated gastric acid secretion. As might be expected at this stage of its development, published therapeutic experience with nizatidine is limited. Nevertheless, multicentre therapeutic trials have shown that nizatidine 300mg at bedtime or 150mg twice daily is significantly more effective than placebo for healing active duodenal ulcer, and is apparently as effective as standard doses of ranitidine in increasing the rate of healing of both duodenal and gastric ulcers, and as effective as a standard dose of cimetidine in active duodenal ulcer. When used prophylactically a single 150mg dose of nizatidine at night produces a decrease in the incidence of ulcer recurrence compared with placebo, and a similar rate of decrease to that achieved with ranitidine 150mg. Nizatidine is well tolerated. Unlike cimetidine it does not have any antiandrogenic effects or alter the hepatic metabolism of drugs. However, only wider clinical experience with nizatidine can accurately determine its relative efficacy and tolerability compared with other antiulcer therapy. Thus, early clinical experience suggests that nizatidine is a useful alternative to the histamine H2-receptor antagonists presently in clinical use.

The enterohepatic circulation of perhexiline metabolites in the male Wistar rat.

1. The biliary excretion of some perhexiline metabolites has been assessed in male Wistar rats with biliary cannulation. 2. After intragastric administration of perhexiline maleate (2 mg/kg body weight) multiple perhexiline metabolites were detected in bile. 3. When aliquots of this metabolite-laden bile were administered intraduoduodenally to further 'recipient' rats with biliary cannulation, similar metabolites were detected in the bile of these rats, but at reduced concentrations equivalent to 30-35% of those present in the bile of 'donor' rats. 4. These findings indicate that in the male Wistar rat, there may be substantial enterohepatic circulation of some perhexiline metabolites.

The pharmacokinetics of proguanil in human subjects following a single oral dose.

The pharmacokinetics of orally administered 200-mg dose of proguanil in volunteers, one African and one Caucasian, is described. The drug was rapidly absorbed reaching a peak concentration in the blood within 3 h, and declining slowly thereafter to give a terminal phase elimination half life of 11.20 +/- 4.10 h and a systemic clearance of 1.270 +/- 0.020 l/h/kg. The small apparent volume of distribution shows that the drug is confined mainly to the blood and is not extensively bound to tissues; it undergoes cyclic oxidation in the liver to cycloguanil--the active metabolite responsible for antimalarial activity. Cycloguanil was detected in the plasma 3 h after proguanil ingestion and reached peak concentration between 5 h and 6 h. Excretion of proguanil was rapid, 60% of the single dose passing through the renal system within 24 h.

Studies on the metabolism of perhexiline in man.

We have studied the disposition of perhexiline and its two major metabolites, M1 and M3, in healthy volunteers and in patients with biliary T-tube drains after cholecystectomy. In healthy volunteers the genetic control for impaired hepatic oxidation is identical for debrisoquine, sparteine, and perhexiline. Poor metabolizers demonstrate markedly reduced production and excretion of the major metabolite, M1. Their production of M3 is also reduced, but to a lesser degree than for M1, confirming substrate stereoselectivity by hepatic oxidases. Biphasic urinary elimination of M1 and M3 is seen in intact extensive oxidizers, whereas only the first phase is apparent in patients with biliary T-tube drainage. This suggests the possibility of enterohepatic recycling of these compounds, which may account for their prolonged elimination. More than 90% of an ingested dose of perhexiline maleate remains unaccounted for at 24 h after ingestion, even in extensive metabolizers. A careful, radiolabelled tissue-distribution study is warranted to elucidate the complicated metabolic fate of perhexiline.

Simultaneous determination of perhexiline and its monohydroxy metabolites in biological fluids by gas chromatography-electron-capture detection.

A rapid and sensitive method for the simultaneous determination of perhexiline and its cis-4-axial and trans-4-equatorial monohydroxy metabolites (M1 and M3, respectively) in human plasma, urine and bile is described. The assay utilises a single diethyl ether extraction, heptafluorobutyric acid anhydride derivatisation and separation and detection by gas chromatography-electron-capture detection. The limits of detection are 0.1 microgram/ml for perhexiline and 0.025 microgram/ml for the M1 and M3 metabolites. This method has been used in a five-day kinetic study of three healthy adult males who ingested a single 300-mg dose of perhexiline maleate. One of these volunteer subjects exhibited elevated plasma perhexiline and markedly reduced plasma and urinary M1 concentrations together with profoundly prolonged plasma and urinary M1 elimination times when compared with the other two subjects. These differences are thought to be of genetic origin. There were also obvious differences in urinary M3 concentrations which were discussed.

Tularemia and rhabdomyolysis.

Rhabdomyolysis as a complication of tularemia occurred in four patients in Tennessee over a four-year period. All patients were severely ill and had renal failure. Tularemia was not suspected initially in any of these patients. One patient died of complications of rhabdomyolysis. At autopsy, multiple abscesses were present within skeletal muscle, suggesting that rhabdomyolysis resulted from direct muscle involvement with Francisella tularensis. Tularemia should be considered in the differential diagnosis of patients with an acute febrile illness and rhabdomyolysis. Conversely, in patients with acute tularemia, the possibility of rhabdomyolysis should be entertained early, especially in those with evidence of myoglobinuria and/or impaired renal function.