Metabolic control of tobacco pollination by sugars and invertases

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrate

Metabolic control of tobacco pollination by sugars and invertases

Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates.Peer Reviewe

Anther-specific carbohydrate supply and restoration of metabolically engineered male sterility

Male-sterile plants are used in hybrid breeding as well as for gene confinement for genetically modified plants in field trials and agricultural production. Apart from naturally occurring mutations leading to male sterility, biotechnology has added new possibilities for obtaining male-sterile plants, although so far only one system is used in practical breeding due to limitations in propagating male-sterile plants without segregations in the next generation or insufficient restoration of fertility when fruits or seeds are to be harvested from the hybrid varieties. Here a novel mechanism of restoration for male sterility is presented that has been achieved by interference with extracellular invertase activity, which is normally specifically expressed in the anthers to supply the developing microspores with carbohydrates. Microspores are symplastically isolated in the locular space of the anthers, and thus an unloading pathway of assimilates via the apoplasmic space is mandatory for proper development of pollen. Antisense repression of the anther-specific cell wall invertase or interference with invertase activity by expressing a proteinacious inhibitor under the control of the anther-specific invertase promoter results in a block during early stages of pollen development, thus causing male sterility without having any pleiotropic effects. Restoration of fertility was successfully achieved by substituting the down-regulated endogenous plant invertase activity by a yeast invertase fused to the N-terminal portion of potato-derived vacuolar protein proteinase II (PiII–ScSuc2), under control of the orthologous anther-specific invertase promoter Nin88 from tobacco. The chimeric fusion PiII–ScSuc2 is known to be N-glycosylated and efficiently secreted from plant cells, leading to its apoplastic location. Furthermore, the Nin88::PiII-ScSuc2 fusion does not show effects on pollen development in the wild-type background. Thus, such plants can be used as paternal parents of a hybrid variety, thereby the introgression of Nin88::PiII-ScSuc2 to the hybrid is obtained and fertility is restored. In order to broaden the applicability of this male sterility/restoration system to other plant species, a phylogenic analysis of plant invertases(β-fructofuranosidases) and related genes of different species was carried out. This reveals a specific clustering of the cell wall invertases with anther-specific expression for dicotyl species and another cluster for monocotyl plants. Thus, in both groups of plants, there seems to be a kind of co-evolution, but no recent common ancestor of these members of the gene family. These findings provide a helpful orientation to classify corresponding candidate genes in further plant species, in addition to the species analysed so far (Arabidopsis, tobacco, tomato, potato, carrots, rice, and wheat)

Development and psychometric evaluation of a new patient -reported outcome measure for stroke self -management: The Southampton Stroke Self - Management Questionnaire (SSSMQ).

BACKGROUND: Self-management is important to the recovery and quality of life of people following stroke. Many interventions to support self-management following stroke have been developed, however to date no reliable and valid outcome measure exists to support their evaluation. This study outlines the development and preliminary investigation of the psychometric performance of a newly developed patient-reported outcome measure (PROM) of self-management competency following stroke; the Southampton Stroke Self-Management Questionnaire (SSSMQ). METHODS: A convenience sample of 87 people who had had a stroke completed responses to the SSSMQ, the Stroke Self-Efficacy Questionnaire and the Stroke Impact Scale. Scaling properties were assessed using Mokken Scale Analysis. Reliability and construct validity were assessed using intra-class correlation coefficient (ICC), Mokken and Cronbach's reliability coefficients and Spearman rank order correlations with relevant measures. RESULTS: Mokken scaling refined the SSSMQ to 28 scalable items. Internal consistency reliability (Mokken r = 0.89) and test-retest reliability (ICC = 0.928) were excellent. Hypotheses of expected correlations with additional measures held, demonstrating good evidence for construct validity. CONCLUSIONS: Early findings suggest the Southampton Stroke Self-Management Questionnaire is a reliable and valid scale of self-management competency. The SSSMQ represents a potentially valid PROM for the evaluation of self-management following stroke

A comparison of once-daily extended-release methylphenidate formulations in children with attention-deficit/hyperactivity disorder in the laboratory school (The Comacs Study)

The objective of this study was to evaluate differences in the pharmacodynamic (PD) profile of 2 second-generation extended-release (ER) formulations of methylphenidate (MPH): Metadate CD (MCD; methylphenidate HCl, US Pharmacopeia) extended-release capsules, CII, and Concerta (CON; methylphenidate HCl) extended-release tablets, CII. Little empirical information exists to help the clinician compare the PD effects of the available ER formulations on attention and behavior. Previous studies have shown that the near-equal doses of MCD and CON provide equivalent, total exposure to MPH as measured by area under the plasma concentration time curve, yet their pharmacokinetic (PK) plasma concentration versus time profiles are different. We previously offered a theoretical PK/PD account of the similarities and differences among available ER formulations based on the hypothesis that all formulations produce effects related to MPH delivered by 2 processes: 1) an initial bolus dose of immediate-release (IR) MPH that is expected to achieve peak plasma concentration in the early morning and have rapid onset of efficacy within 2 hours of dosing, which for the MCD capsule is delivered by 30% of the total daily dose as uncoated beads and for the CON tablet is delivered by an overcoat of 22% of the total daily dose; and 2) an extended, controlled delivery of ER MPH that is expected to achieve peak plasma concentrations in the afternoon to maintain efficacy for a programmed period of time after the peak of the initial bolus, which for the MCD capsule is delivered by polymer-coated beads and for the CON tablet by an osmotic-release oral system. According to this PK/PD model, clinical superiority is expected at any point in time for the formulation with the highest MPH plasma concentration.Methods This was a multisite, double-blind, double-dummy, 3-way crossover study of 2 active treatments (MCD and CON) and placebo (PLA). Children with confirmed diagnoses of attention-deficit/hyperactivity disorder were stratified to receive bioequivalent doses of MCD and CON that were considered to be low (20 mg of MCD and 18 mg of CON), medium (40 mg of MCD and 36 mg of CON), or high (60 mg of MCD and 54 mg of CON), and in a randomized order each of the study treatments was administered once daily in the morning for 1 week. On the seventh day of each treatment week, children attended a laboratory school, where surrogate measures of response were obtained by using teacher ratings of attention and deportment and a record of permanent product of performance on a 10-minute math test at each of the 7 classroom sessions spread across the day at 1.5-hour intervals. Safety was assessed by patient reports of adverse events, parent ratings on a stimulant side-effects scale, and measurement of vital signs.Results The analyses of variance revealed large, statistically significant main effects for the within-subject factor of treatment for all 3 outcome measures (deportment, attention, and permanent product). The interactions of treatment x session were also highly significant for all 3 outcome measures. Inspection of the PD profiles for the treatment x session interactions suggested 4 patterns of efficacy across the day: 1) PLA &gt; MCD CON (PLA superiority) immediately after dosing; 2) MCD &gt; CON &gt; PLA during the morning (MCD superiority); 3) MCD CON &gt; PLA during the afternoon (PD equivalence of MCD and CON); and 4) CON &gt; MCD PLA in the early evening (CON superiority). The effect of site was significant, because some study centers had low and some high scores for behavior in the lab classroom, but both the low- and high-scoring sites showed similar PD patterns across the day. The interaction of dose x treatment was not significant, indicating that the pattern of treatment effects was consistent across each dose level. There were no statistically significant overall differences among the 3 treatments for the frequency of treatment-emergent adverse events, ratings of side effects, or vital signs. Two additional PK/PD questions were addressed: 1. The a priori hypothesis called for a comparison of the average of sessions (removing session as a factor) during a time period that corresponds to the length of a typical school day (from 1.5 through 7.5 hours after dosing). For the planned contrast of the 2 treatment conditions (MCD versus CON), the difference was significant, confirming the a priori hypothesis of superiority of near-equal daily doses of MCD over CON for this predefined postdosing period. 2. In the design of the study, the dose factor represented the total daily dose, consisting of 2 components: the initial bolus doses of IR MPH, which differ for the near-equal total daily doses of MCD and CON, and the reservoir doses of ER MPH, which were the same for the 2 formulations. To evaluate the moderating effects of the bolus component of dose on outcome, average effect size (ES) was calculated for the efficacy outcomes at the time of expected peak PK concentration times of the initial bolus component for each formulation at the 3 dose levels. The correlation (r) of ES with IR MPH bolus dose was significant for each of the 3 outcome measures (r .9), indicating that the magnitude of effects in the early morning may be attributed to the dose administered by the IR MPH bolus of each formulation. For the 2 dose conditions with equal 12-mg IR MPH boluses (MCD 40 and CON 54), the ESs were large and indistinguishable (eg, deportment ES 0.75 for both).Conclusions Once-daily doses of MCD and CON produced statistically significantly different PD effects on surrogate measures of behavior and performance among children with attention-deficit/hyperactivity disorder in the laboratory school setting. As predicted by the PK/PD model, superiority at any point in time was achieved by the formulation with the highest expected plasma MPH concentration.<br/