Integrating Rapid Phenotyping and Speed Breeding to Improve Stay-Green and Root Adaptation of Wheat in Changing, Water-Limited, Australian Environments

Temperatures have increased and in-crop rainfall decreased over recent decades in many parts of the Australian wheat cropping region. With these trends set to continue or intensify, improving crop adaptation in the face of climate change is particularly urgent in this, already drought-prone, cropping region. Importantly, improved performance under water-limitation must be achieved while retaining yield potential during more favourable seasons. A multi-trait-based approach to improve wheat yield and yield stability in the face of water-limitation and heat has been instigated in northern Australia using novel phenotyping techniques and a nested association mapping (NAM) approach. An innovative laboratory technique allows rapid root trait screening of hundreds of lines. Using soil grown seedlings, the method offers significant advantages over many other lab-based techniques. Another recently developed method allows novel stay-green traits to be quantified objectively for hundreds of genotypes in standard field trial plots. Field trials in multiple locations and seasons allow evaluation of targeted trait values and identification of superior germplasm. Traits, including yield and yield components are measured for hundreds of NAM lines in rain fed environments under various levels of water-limitation. To rapidly generate lines of interest, the University of Queensland “speed breeding” method is being employed, allowing up to 7 plant generations per annum. A NAM population of over 1000 wheat recombinant inbred lines has been progressed to the F5 generation within 18 months. Genotyping the NAM lines with the genome-wide DArTseq molecular marker system provides up to 40,000 markers. They are now being used for association mapping to validate QTL previously identified in bi-parental populations and to identify novel QTL for stay-green and root traits. We believe that combining the latest techniques in physiology, phenotyping, genetics and breeding will increase genetic progress toward improved adaptation to water-limited environments.

Effects of oxygen provision on the physiology of baker's yeast Saccharomyces cerevisiae

The availability of oxygen has a major effect on all organisms. The yeast Saccharomyces cerevisiae is able to adapt its metabolism for growth in different conditions of oxygen provision, and to grow even under complete lack of oxygen. Although the physiology of S. cerevisiae has mainly been studied under fully aerobic and anaerobic conditions, less is known of metabolism under oxygen-limited conditions and of the adaptation to changing conditions of oxygen provision. This study compared the physiology of S. cerevisiae in conditions of five levels of oxygen provision (0, 0.5, 1.0, 2.8 and 20.9% O2 in feed gas) by using measurements on metabolite, transcriptome and proteome levels. On the transcriptional level, the main differences were observed between the three level groups, 0, 0.5 2.8 and 20.9% O2 which led to fully fermentative, respiro-fermentative and fully respiratory modes of metabolism, respectively. However, proteome analysis suggested post-transcriptional regulation at the level of 0.5 O2. The analysis of metabolite and transcript levels of central carbon metabolism also suggested post-transcriptional regulation especially in glycolysis. Further, a global upregulation of genes related to respiratory pathways was observed in the oxygen-limited conditions and the same trend was seen in the proteome analysis and in the activities of enzymes of the TCA cycle. The responses of intracellular metabolites related to central carbon metabolism and transcriptional responses to change in oxygen availability were studied. As a response to sudden oxygen depletion, concentrations of the metabolites of central carbon metabolism responded faster than the corresponding levels of gene expression. In general, the genome-wide transcriptional responses to oxygen depletion were highly similar when two different initial conditions of oxygen provision (20.9 and 1.0% O2) were compared. The genes related to growth and cell proliferation were transiently downregulated whereas the genes related to protein degradation and phosphate uptake were transiently upregulated. In the cultures initially receiving 1.0% O2, a transient upregulation of genes related to fatty acid oxidation, peroxisomal biogenesis, response to oxidative stress and pentose phosphate pathway was observed. Additionally, this work analysed the effect of oxygen on transcription of genes belonging to the hexose transporter gene family. Although the specific glucose uptake rate was highest in fully anaerobic conditions, none of the hxt genes showed highest expression in anaerobic conditions. However, the expression of genes encoding the moderately low affinity transporters decreased with the decreasing oxygen level. Thus it was concluded that there is a relative increase in high affinity transport in anaerobic conditions supporting the high uptake rate.

Spectral Tuning and Adaptation to Different Light Environments of Mysid Visual Pigments

In the present thesis, questions of spectral tuning, the relation of spectral and thermal properties of visual pigments, and evolutionary adaptation to different light environments were addressed using a group of small crustaceans of the genus Mysis as a model. The study was based on microspectrophotometric measurements of visual pigment absorbance spectra, electrophysiological measurements of spectral sensitivities of dark-adapted eyes, and sequencing of the opsin gene retrieved through PCR. The spectral properties were related to the spectral transmission of the respective light environments, as well as to the phylogentic histories of the species. The photoactivation energy (Ea) was estimated from temperature effects on spectral sensitivity in the long-wavelength range, and calculations were made for optimal quantum catch and optimal signal-to-noise ratio in the different light environments. The opsin amino acid sequences of spectrally characterized individuals were compared to find candidate residues for spectral tuning. The general purpose was to clarify to what extent and on what time scale adaptive evolution has driven the functional properties of (mysid) visual pigments towards optimal performance in different light environments. An ultimate goal was to find the molecular mechanisms underlying the spectral tuning and to understand the balance between evolutionary adaptation and molecular constraints. The totally consistent segregation of absorption maxima (λmax) into (shorter-wavelength) marine and (longer-wavelength) freshwater populations suggests that truly adaptive evolution is involved in tuning the visual pigment for optimal performance, driven by selection for high absolute visual sensitivity. On the other hand, the similarity in λmax and opsin sequence between several populations of freshwater M. relicta in spectrally different lakes highlights the limits to adaptation set by evolutionary history and time. A strong inverse correlation between Ea and λmax was found among all visual pigments studied in these respects, including those of M. relicta and 10 species of vertebrate pigments, and this was used to infer thermal noise. The conceptual signal-to-noise ratios thus calculated for pigments with different λmax in the Baltic Sea and Lake Pääjärvi light environments supported the notion that spectral adaptation works towards maximizing the signal-to-noise ratio rather than quantum catch as such. Judged by the shape of absorbance spectra, the visual pigments of all populations of M. relicta and M. salemaai used exclusively the A2 chromophore (3, 4-dehydroretinal). A comparison of amino acid substitutions between M. relicta and M. salemaai indicated that mysid shrimps have a small number of readily available tuning sites to shift between a shorter - and a longer -wavelength opsin. However, phylogenetic history seems to have prevented marine M. relicta from converting back to the (presumably) ancestral opsin form, and thus the more recent reinvention of marine spectral sensitivity has been accomplished by some other novel mechanism, yet to be found

Physiological Determinants of Invasive Success Linking Distribution Patterns to Metabolic Physiology in Native and Invasive Blue Mussels (genus Mytilus)

In the early 20th century, a blue mussel species from the Mediterranean invaded the California coast and subsequently out-competed the native species south of Monterey Bay. Like other invasive species, Mytilus galloprovincialis has physiological traits that make it successful in habitats formerly occupied by the native M. trossulus, namely its adaptation to warm sea surface temperatures. This study looks at the current genotype distributions and enzymatic activities of field-acclimatized mussels within the hybrid zone where the species co-occur as well as mussels that have been acclimated for four weeks to different temperature and salinity conditions. In the field-acclimatized and laboratory-acclimated mussels, the native species exhibited significantly higher enzyme rates, which may reflect an evolutionary adaptation to compensate to low habitat temperatures. Indeed, the results of the laboratory acclimation indicate that these differences are genetically based. Whether an acclimation capacity exists may require even longer-term acclimation to different temperatures. Current findings suggest that the further spread of the invasive species is likely to be governed in large measure by the potentially counteracting effects of rising temperatures, which would favor the northerly spread of M. galloprovincialis, and increased winter precipitation, which would favor the persistence of M. trossulus. However, the success of M. galloprovincialis during acclimation to ‘dilute’ salinity (25 ppt) suggests that the invasive species can tolerate a greater salinity range than previously thought. Thus, further investigation is needed to build a comprehensive predictive model of the movement of M. galloprovincialis and the hybrid zone along the California coast.

Physiological Adaptation Of Mytilus-Edulis To Cyclic Temperatures

Mytilus edulis adapted to cyclic temperatures by reducing the amplitude of response of oxygen consumption and filtration rate over a period of approximately two weeks, and thereby increasing their independence of temperature within the range of the fluctuating regime. When acclimated to cyclic temperature regimes within the range from 6 to 20°C, the metabolic and feeding rates, measured at different temperatures in the cycle, were not significantly different from the adapted response to equivalent constant temperatures. Physiological adaptation ofMytilus edulis to different thermal environments was reflected in their metabolic and feeding rate-temperature curves. Animals subjected to marked diel fluctuations in environmental temperature showed an appropriate region of temperature-independence, whereas animals from a population not experiencing large diel temperature fluctuations showed no region of temperature-independence. In a fluctuating thermal environment which extended above the normal environmental maxima, respiratory adaptation occurred at higher temperatures than was possible in a constant thermal environment. The feeding rate was also maintained at higher temperatures in a cyclic regime than was possible under constant thermal conditions. This represented a shortterm extension of the zone of activity in a fluctuating thermal environment. The net result of these physiological responses to high cyclic and constant temperatures has been assessed in terms of ‘scope for growth’. Animals acclimated to cyclic temperatures between 21 and 29°C had a higher scope for growth at 29°C and were less severely stressed than those maintained at the constant temperature of 29°C.

Neuromuscular adaptation during skill acquisition on a two degree-of-freedom target-acquisition task: Isometric torque production

In this study we attempted to identify the principles that govern the changes in neural control that occur during repeated performance of a multiarticular coordination task. Eight participants produced isometric flexion/extension and pronation/supination torques at the radiohumeral joint, either in isolation (e.g., flexion) or in combination (e.g., flexion - supination), to acquire targets presented by a visual display. A cursor superimposed on the display provided feedback of the applied torques. During pre- and postpractice tests, the participants acquired targets in eight directions located either 3.6 cm (20% maximal voluntary contraction [MVC]) or 7.2 cm (40% MVC) from a neutral cursor position. On each of five consecutive days of practice the participants acquired targets located 5.4 cm (30% MVC) from the neutral position. EMG was recorded from eight muscles contributing to torque production about the radiohumeral joint during the pre- and posttests. Target-acquisition time decreased significantly with practice in most target directions and at both target torque levels. These performance improvements were primarily associated with increases in the peak rate of torque development after practice. At a muscular level, these changes were brought about by increases in the rates of recruitment of all agonist muscles. The spatiotemporal organization of muscle synergies was not significantly altered after practice. The observed adaptations appear to lead to performances that are generalizable to actions that require both greater and smaller joint torques than that practiced, and may be successfully recalled after a substantial period without practice. These results suggest that tasks in which performance is improved by increasing the rate of muscle activation, and thus the rate of joint torque development, may benefit in terms of the extent to which acquired levels of performance are maintained over time.

Neuromuscular adaptation during skill acquisition on a two degree-of-freedom target-acquisition task: Dynamic movement

In this experiment, we examined the extent to which the spatiotemporal reorganization of muscle synergies mediates skill acquisition on a two degree-of-freedom (df) target-acquisition task. Eight participants completed five practice sessions on consecutive days. During each session they practiced movements to eight target positions presented by a visual display. The movements required combinations of flexion/extension and pronation/supination of the elbow joint complex. During practice sessions, eight targets displaced 5.4 cm from the start position ( representing joint excursions of 54) were presented 16 times. During pre- and posttests, participants acquired the targets at two distances (3.6 cm [36 degrees] and 7.2 cm [72 degrees]). EMG data were recorded from eight muscles contributing to the movements during the pre- and posttests. Most targets were acquired more rapidly after the practice period. Performance improvements were, in most target directions, accompanied by increases in the smoothness of the movement trajectories. When target acquisition required movement in both dfs, there were also practice-related decreases in the extent to which the trajectories deviated from a direct path to the target. The contribution of monofunctional muscles ( those producing torque in a single df) increased with practice during movements in which they acted as agonists. The activity in bifunctional muscles ( those contributing torque in both dfs) remained at pretest levels in most movements. The results suggest that performance gains were mediated primarily by changes in the spatial organization of muscles synergies. These changes were expressed most prominently in terms of the magnitude of activation of the monofunctional muscles.

The sites of neural adaptation induced by resistance training in humans

Although it has long been supposed that resistance training causes adaptive changes in the CNS, the sites and nature of these adaptations have not previously been identified. In order to determine whether the neural adaptations to resistance training occur to a greater extent at cortical or subcortical sites in the CNS, we compared the effects of resistance training on the electromyographic (EMG) responses to transcranial magnetic (TMS) and electrical (TES) stimulation. Motor evoked potentials (MEPs) were recorded from the first dorsal interosseous muscle of 16 individuals before and after 4 weeks of resistance training for the index finger abductors (n=8), or training involving finger abduction-adduction without external resistance (n=8). TMS was delivered at rest at intensities from 5% below the passive threshold to the maximal output of the stimulator. TMS and TES were also delivered at the active threshold intensity while the participants exerted torques ranging from 5 to 60% of their maximum voluntary contraction (MVC) torque. The average latency of MEPs elicited by TES was significantly shorter than that of TMS MEPs (TES latency=21.5+/-1.4 ms; TMS latency=23.4+/-1.4 ms; P

The Synergistic Organization of Muscle Recruitment Constrains Visuomotor Adaptation

Reaching to visual targets engages the nervous system in a series of transformations between sensory information and motor commands. That which remains to be determined is the extent to which the processes that mediate sensorimotor adaptation to novel environments engage neural circuits that represent the required movement in joint-based or muscle-based coordinate systems. We sought to establish the contribution of these alternative representations to the process of visuomotor adaptation. To do so we applied a visuomotor rotation during a center-out isometric torque production task that involved flexion/extension and supination/pronation at the elbow-joint complex. In separate sessions, distinct half-quadrant rotations (i.e., 45°) were applied such that adaptation could be achieved either by only rescaling the individual joint torques (i.e., the visual target and torque target remained in the same quadrant) or by additionally requiring torque reversal at a contributing joint (i.e., the visual target and torque target were in different quadrants). Analysis of the time course of directional errors revealed that the degree of adaptation was lower (by ~20%) when reversals in the direction of joint torques were required. It has been established previously that in this task space, a transition between supination and pronation requires the engagement of a different set of muscle synergists, whereas in a transition between flexion and extension no such change is required. The additional observation that the initial level of adaptation was lower and the subsequent aftereffects were smaller, for trials that involved a pronation–supination transition than for those that involved a flexion–extension transition, supports the conclusion that the process of adaptation engaged, at least in part, neural circuits that represent the required motor output in a muscle-based coordinate system.

Adaptation and Reintegration of Proprioceptive Information in Young and Older Adults' Postural Control

We investigated age-related changes in adaptation and sensory reintegration in postural control without vision. In two sessions, participants adapted their posture to sway reference and to reverse sway reference conditions, the former reducing (near eliminating) and the latter enhancing (near doubling) proprioceptive information for posture by means of support-surface rotations in proportion to body sway. Participants stood on a stable platform for 3 min (baseline) followed by 18 min of sway reference or reverse sway reference (adaptation) and finally again on a stable platform for 3 min (reintegration). Results showed that when inaccurate proprioception was introduced, anterior-posterior (AP) sway path length increased in comparable levels in the two age groups. During adaptation, young and older adults reduced postural sway at the same rate. On restoration of the stable platform in the reintegration phase, a sizeable aftereffect of increased AP path length was observed in both groups, which was greater in magnitude and duration for older adults. In line with linear feedback models of postural control, spectral analyses showed that this aftereffect differed between the two platform conditions. In the sway-referenced condition, a switch from low- to high-frequency COP sway marked the transition from reduced to normal proprioceptive information. The opposite switch was observed in the reverse sway referenced condition. Our findings illustrate age-related slowing in participants' postural control adjustments to sudden changes in environmental conditions. Over and above differences in postural control, our results implicate sensory reweighting as a specific mechanism highly sensitive to age-related decline.

Polymorphism and physiology of arsenate tolerance in Holcus lanatus L. from an uncontaminated site

The polymorphism of arsenate tolerance in a Holcus lanatus L. population from an uncontaminated soil was investigated and a high percentage of tolerant individuals (65%) was found in the population studied. Influx of arsenate was highly correlated to arsenate tolerance within the population, with the most tolerant individuals having the lowest rates of arsenate influx. Isotherms for the high affinity arsenate uptake systems were determined in six tolerant and six non-tolerant genotypes. Tolerant plants had the lowest rates of arsenate influx. This was achieved by adaptation of the V_max of arsenate influx with the V_max of the high affinity uptake system saturating at lower substrate concentrations in the tolerant plants. The polymorphism is discussed with relation to adaptation to the extreme environments to which the plants are subjected on mine-spoil soils. © 1992 Kluwer Academic Publishers.

A possible role for intrinsic calcium buffers in the long- term adaptation of crayfish neurons

Increasing the impulse activity of neurons in vivo over 3 or more days causes a reduction in transmitter release that persists for days or weeks (eg. Mercier and Atwood, 1989). This effect is usually accompanied by decreased synaptic fatigue. These two changes involve presynaptic mechanisms and indicate "long-term adaptation" (LTA) of nerve terminals. Previous experiments have shown that LTA requires extracellular calcium and protein synthesis (eg. Hong and Lnenicka, Soc. Neurosci. Abstr. 17:1322) and appears to involve communication between the cell body and the nerve terminals. The present study examines the possibility that the reduction in transmitter release is caused by an -increase in the calcium buffering ability within the nerve terminals. It examines the responses of adapted and control nerve terminals to exogenously applied calcium buffer, BAPTA-AM, which decreases transmitter release (Robitialle and Charlton, 1992). If LTA increases intrinsic Ca2+-buffering, the membrane permeant form of BAPTA should have less effect on adapted nerve terminals than on controls. Experiments are performed on the phasic abdominal extensor motor neurons of the crayfish, Procambarns clarkii. BAPTA-AM decreases excitatory postsynaptic potentials (EPSP's) of the phasic extensor muscles in a dosedependent manner between 5 and 50 JLM. LTA is elicited by in vivo stimulation at 2.5 Hz for 2-4 h per day over 3 days, which reduces EPSP's by over 50%. Experiments indicate that BAPTA-AM produces no significant change in EPSP reduction in adapted neurons when compared to controls. These results do not support the hypothesis that increased daily activity alters rapid intrinsic calcium buffers, that are able to reduce transmitter output in the same manner as BAPTA.

Dietary copper absorption and excretion in three semi-terrestrial grapsoid crabs with different levels of terrestrial adaptation

Three species of phylogenetically related semi-terrestrial crabs (Superfamily Grapsoidea - Sesarma rectum, Goniopsis cruentata and Neohelice granulata (formerly: Chasmagnathus granulatus) with different degrees of terrestriality were studied to quantify the accumulation of copper (Cu) in hemolymph, gills, hepatopancreas and antennal gland, and its excretion through the faeces. These crabs were fed for 15 days practical diets containing 0 (A), 0.5 (B), 1.0 (C), and 1.5% (D) of added CuCl2 (corresponding to 0, 0.2, 0.5 and 0.7% of Cu2+, respectively). The amount of food ingested was directly proportional to the degree of terrestriality: S. rectum, the most terrestrial species, ate around 2-3 times more than the other crabs, whereas G. cruentata ate 1.5-2 times more than N. granulata, the least terrestrial. The amount of Cu excreted in the feces was proportional to Cu ingestion, and was 76.8% and 64.2% higher for Sesarma fed diet D compared to G. cruentata and N. granulata, respectively. Sesarma also displayed higher Cu concentration in the haemolymph, gills and antennal glands, but not in the hepatopancreas. A detoxifying mechanism followed by elimination was probably present at this last organ, preventing Cu accumulation. More terrestrial crabs, such as Sesarma, may accumulate more Cu in hemolymph and tissues, showing a correlation between metal accumulation and increased terrestriality. In this aspect, contaminated feed sources with Cu may have more impact in conservation of terrestrial crabs. (C) 2008 Elsevier Inc. All rights reserved.

INSECT CHYMOTRYPSINS: CHLOROMETHYL KETONE INACTIVATION AND SUBSTRATE SPECIFICITY RELATIVE TO POSSIBLE COEVOLUTIONAL ADAPTATION OF INSECTS AND PLANTS

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Ewer insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization. using different, colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from, bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe P-nitroanilide). Chloromethyl ketones (TPCK, N-alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N-carbobenzoxy-Gly-Gly-phe-CK) inactivated all chymotrypsins legated. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1)s(-1); Z-GGF-CK, 150 to 450 M(-1)s(-1) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1)s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that. in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa, value. This is Proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed. to be an adaptation to the presence of dietary ketones. (C) 2009 Wiley Periodicals, Inc.

Dietary copper absorption and excretion in three semi-terrestrial grapsoid crabs with different levels of terrestrial adaptation

Three species of phylogenetically related semi-terrestrial crabs (Superfamily Grapsoidea - Sesarma rectum, Goniopsis cruentata and Neohelice granulata (formerly: Chasmagnathus granulatus) with different degrees of terrestriality were studied to quantify the accumulation of copper (Cu) in hemolymph, gills, hepatopancreas and antennal gland, and its excretion through the faeces. These crabs were fed for 15 days practical diets containing 0 (A), 0.5 (B), 1.0 (C), and 1.5% (D) of added CuCl2 (corresponding to 0, 0.2, 0.5 and 0.7% of Cu2+, respectively). The amount of food ingested was directly proportional to the degree of terrestriality: S. rectum, the most terrestrial species, ate around 2-3 times more than the other crabs, whereas G. cruentata ate 1.5-2 times more than N. granulata, the least terrestrial. The amount of Cu excreted in the feces was proportional to Cu ingestion, and was 76.8% and 64.2% higher for Sesarma fed diet D compared to G. cruentata and N. granulata, respectively. Sesarma also displayed higher Cu concentration in the haemolymph, gills and antennal glands, but not in the hepatopancreas. A detoxifying mechanism followed by elimination was probably present at this last organ, preventing Cu accumulation. More terrestrial crabs, such as Sesarma, may accumulate more Cu in hemolymph and tissues, showing a correlation between metal accumulation and increased terrestriality. In this aspect, contaminated feed sources with Cu may have more impact in conservation of terrestrial crabs. (C) 2008 Elsevier B.V. All rights reserved.