DNA damage formation and p53 accumulation in mammalian cells exposed to the space environment.

To determine the effects of the space environment on gene instability from the point of view of human health for long-term stays in space, we have studied the formation of DNA strand breaks and the induction of gene expression in mammalian cells. We previously measured DNA damage in human cultured cells and the accumulation of a tumor suppressor gene product, p53, in muscle and skin of rats after space flight, and the relative importance of microgravity and space radiation in causing these effects remains to be clarified. Our results suggest that the p53 pathway may play a role in safeguarding genomic stability against the stressful space environment. We review here the present knowledge on cellular stress signaling and present our space experimental data. The importance of the stress response to the space environment is also discussed.     

The biophysical limitations in physiological transport and exchange in plants grown in microgravity

This paper describes how changes in the physical behavior of fluids and gases in microgravity can limit the physiological transport and exchange in higher plants. These types of effects are termed indirect effects of microgravity because they are not due to gravity interacting with the mass of the plant body itself. The impact of limiting gravity-dependent transport phenomena has been analyzed by the use of mathematical modeling to simulate and compare biophysical transport in the 1g and spaceflight environments. These data clearly show that the microgravity environment induces significant limitations on basic physiological and biochemical processes within the aerial and rootzone portions of the plant. Furthermore, this mathematical model provides a solid foundation for explaining the physiological effects that have been noted in past spaceflight experiments.     

Artificial gravity in space and in medical research

The history of manned space flight has repeatedly documented the fact that prolonged sojourn in space causes physiological deconditioning. Physiological deterioration has raised a legitimate concern about man's ability to adequately perform in the course of long missions and even the possibility of leading to circumstances threatening survival. One of the possible countermeasures of physiological deconditioning, theoretically more complete than others presently used since it affects all bodily systems, is artificial gravity. Space stations and spacecrafts can be equipped with artificial gravity, but is artificial gravity necessary? The term "necessary" must be qualified because a meaningful answer to the question depends entirely on further defining the purpose of space travel. If man intends to stay only temporarily in space, then he must keep himself in good physical condition so as to be able to return to earth or to land on any other planetary surface without undue exposure to major physiological problems resulting from transition through variable gravitational fields. Such a situation makes artificial gravity highly desirable, although perhaps not absolutely necessary in the case of relative short exposure to microgravity, but certainly necessary in interplanetary flight and planetary landings. If the intent is to remain indefinitely in space, to colonize space, then artificial gravity may not be necessary, but in this case the consequences of long term effects of adaptation to weightlessness will have to be weighed against the biological evolutionary outcomes that are to be expected. At the moment, plans for establishing permanent colonies in space seem still remote. More likely, the initial phase of exploration of the uncharted solar system will take place through successive, scope limited, research ventures ending with return to earth. This will require man to be ready to operate in gravitational fields of variable intensity. Equipping spacecrafts or space stations with some means of artificial gravity in this initial phase is, therefore, necessary without question. In a strict sense artificial gravity is conceived as a means of replacing natural gravity in space by the centripetal acceleration generated by some sort of rotating device. Rotating devices create an inertial force which has effects on bodies similar to those caused by terrestrial gravity, but artificial gravity by a rotation device is not the same as terrestrial gravity, as we shall see. Present research in artificial gravity for space exploration is projected in two main directions: artificial gravity for whole space stations and artificial gravity produced by short arm centrifuges designed for human use in space.     

Drought stress, plant water status, and floral trait expression in fireweed, Epilobium angustifolium (Onagraceae)

In a controlled environment, we artificially induced drought during flowering of Epilobium angustifolium, an animal-pollinated plant. Leaf water potential (ψ(l)) and floral traits were monitored over a 12-d period of soil moisture depletion. Soil moisture depletion induced drought stress over time, as revealed by significant treatment × day interactions for predawn and midday ψ(l). Nectar volume and flower size showed significant negative responses to drought stress, but nectar sugar concentration did not vary between treatments. Floral traits were more buffered from drought than leaf water potentials. We used path analysis to examine direct and indirect effects of ψ(l) on floral traits for plants in well-watered (control) vs. drought treatments. According to the best-fit path models, midday ψ(l) has significant positive effects on flower size and nectar volume in both environments. However, for controls midday ψ(l) also had a significant negative effect on nectar sugar concentration. Results indicate that traits influencing floral attractiveness to pollinators in E. angustifolium vary with plant water status, such that pollinator-mediated selection could indirectly target physiological or biochemical controls on ψ(l). Moreover, under mesic conditions selection for greater nectar sugar reward may be constrained by the antagonistic effects of plant water status on nectar volume and sugar concentration.     

Vector-averaged gravity does not alter acetylcholine receptor single channel properties.

To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.     

基于“三生空间”的土地利用功能演变及生态环境响应——以桂西资源富集区为例

分析土地利用功能演变及生态环境响应是区域"三生空间"划定和管控以及生态环境保护的重要依据。以桂西资源富集区为例,基于1995年、2000年、2005年、2010年和2015年5期遥感数据,构建"三生空间"土地利用主导功能分类体系,运用转移矩阵、重心转移和生态环境响应模型分析研究区20年土地利用功能演变特征及生态环境响应情况,结果表明:(1)研究区生态生产空间和生活生产空间面积呈上升趋势,生产生态空间和生态空间面积呈下降趋势。(2)生态生产空间重心转移距离最大,其次是生活生产空间重心,生产生态空间和生态空间重心转移距离较小。(3)研究区生态环境质量指数从1995年的0.6337持续降至2015年的0.6323,整体质量有所下降。(4)以林草地为主要地类的县域生态环境质量较高,重点矿产资源型县域和农产品生产基地生态环境质量较低,城镇化重点县域生态环境质量最低。     

Fitness consequences of cotyledon and mature-leaf damage in the ivyleaf morning glory

To understand the evolutionary and ecological consequences of natural enemy damage to plants, it is essential to determine how the fitness effects of damage differ depending on the tissues damaged and the subsequent pattern of damage. In a field experiment with the ivyleaf morning glory, the direct and indirect effects on fitness of herbivore damage to cotyledons and mature leaves was evaluated. Damage to mature leaves had negligible direct effects on fitness and no indirect effects on fitness through other correlated traits. Damage to cotyledons also did not directly affect fitness, but did so indirectly through its effects on plant size. These findings suggest that increased resistance to cotyledon damage or increased compensatory growth following cotyledon damage could be effective strategies for plants of this species to counteract the negative effects of herbivory.     

Attracting pollinators and avoiding herbivores: insects influence plant traits within and across years

Perennial plants interact with herbivores and pollinators across multiple growing seasons, and thus may respond to herbivores and pollinators both within and across years. Joint effects of herbivores and pollinators influence plant traits, but while some of the potential interactions among herbivory, pollination, plant size, and plant reproductive traits have been well studied, others are poorly understood. This is particularly true for perennial plants where effects of herbivores and pollinators may manifest across years. Here, we describe two experiments addressing the reciprocal interactions of plant traits with herbivore damage and pollination across 2 years using the perennial plant Chamerion angustifolium. We measured (1) plant responses to manipulation of damage and pollination in the year of treatment and the subsequent season, (2) damage and pollination responses to manipulation of plant size and flowering traits in the year of treatment, and (3) plant-mediated indirect interactions between herbivores and pollinators. We found that plant traits had little effect on damage and pollination, but damage and pollination affected plant traits in both the treatment year and the subsequent year. We found evidence of indirect effects between leaf herbivores and pollinators in both directions; indirect effects of pollinators on leaf herbivores have not been previously demonstrated. Our results indicate that pollen receipt results in shorter plants with fewer stems but does not change flower number, while leaf herbivory results in taller plants with fewer flowers. Together, herbivory and pollination may contribute to intermediate plant height and plants with fewer stems and flowers in our system.     

Direct and indirect effects of marsupial browsing on a foundation tree species

Plant‐mediated indirect effects can be important ecological drivers in plant communities, especially in systems where extended genetic effects of foundation species can shape communities and influence ecosystem dynamics. Here we investigate the direct and indirect effects of uncontrolled browsing by marsupial herbivores including the common brushtail possum Trichosurus vulpecula, Bennetts wallaby Macropus rufogriseus and the red‐bellied pademelon Thylogale billardierii, in a Eucalyptus system known to have extended community and ecosystem genetic effects. In a common garden trial containing 525 full‐sib families from an incomplete diallel crossing program located in northeastern Tasmania, Australia, we assessed the genetic basis to herbivore preferences, the impact of a single and repeated marsupial browsing event on tree fitness and morphological traits and the associated indirect plant‐mediated effects on a subsequent herbivore, autumn gum moth Mnesampela privata. Marsupial browsing was not influenced by plant genetics, but spatial components instead affected the pattern of damage across the trial. Marsupial browsing had significant impacts on tree development, morphology and survival, resulting in reductions in survival, height and basal area, an increase proportion in multiple stems, delays in flowering as well as delays in phase change from juvenile to adult foliage. Fitness impacts were minimal in response to a once‐off browsing event, but effects were exacerbated when trees suffered repeated browsing. We demonstrate clear plant‐mediated indirect effects of marsupial browsing on subsequent tree use by an invertebrate herbivore, through induced changes in plant morphology. Such indirect effects have the potential to influence biotic community structure on a foundation species host‐plant, and the evolutionary interactions that occur between organisms and the host‐plant themselves.     

Positive interactions in plant communities and the individualistic-continuum concept

The individualistic nature of communities is held as a fundamental ecological tenet by many ecologists. The empirical rationale for the individualistic hypothesis is largely based on gradient analyses in which plant species are almost always found to be arranged independently of one another in “continua” along environmental gradients. However, continua are correlative patterns and do not identify the processes that determine them, and so they do not necessarily preclude the possibility of interdependent interactions within plant communities. For example, the common occurrence of positive interactions suggests that plant species may not always be distributed independently of each other. If the distributions and abundances of species are enhanced by the presence of other species, their organization is not merely a coincidence of similar adaptation to the abiotic environment. Interpretations of gradient analyses also appear to assume that interactions among species should be similar at all points along environmental axes, and that groups of species should be associated at all points on a gradient if interdependence is to be accepted. However, virtually all types of ecological interactions have been shown to vary with changes in the abiotic environment, and a number of field experiments indicate that positive effects become stronger as abiotic stress increases. Furthermore, interactions among plants have been shown to shift from competition to facilitation along environmental continua. Thus, significant interdependence may occur even when species do not fully overlap in distribution. Higher-order, indirect interactions between animals and plants, and among plants, also suggest that interdependence within communities occurs. Eliminating a species involved in an indirect interaction may not necessarily mean that its beneficiary will be eliminated from a community, but the prospect that the distribution and abundance of any species in a plant community may be positively affected by the effects that other species have on their competitors suggests that communities are organized by much more than “the fluctuating and fortuitous immigration of plants and an equally fluctuating and variable environment” as stated by Henry Gleason. The ubiquity of direct and indirect positive interactions within plant communities provides a strong argument that communities are more interdependent than current theories allow. Received: 17 February 1997 / Accepted: 23 May 1997     

Light environment and the impacts of foliage quality on herbivorous insect attack and bird predation

Theory predicts that variation in plant traits will modify both the direct interactions between plants and herbivores and the indirect impacts of predators of those herbivores. Light has strong effects on leaf quality, so the impacts of herbivores and predators may differ between plants grown in sun and shade. However, past experiments have often been unable to separate the effects of light environment on plant traits and herbivory from direct effects on herbivores and predators. We first manipulated light availability in an open habitat using a shade cloth pre-treatment to produce oak saplings with different leaf qualities. Leaves on plants exposed to high light were thicker and tougher and had lower nitrogen and water contents, and higher carbon and phenolic contents than leaves on plants under a shade cloth. Then, in the main experiment, we moved all plants to a common shade environment where bird predators were excluded in a factorial design. We measured insect herbivore abundance and leaf damage. Herbivores were significantly more abundant and caused greater leaf damage on sun trees, although these leaf characteristics are usually associated with low-quality food. Bird exclusion did not change herbivore abundance but did increase leaf damage. Contrary to our predictions, the effects of birds did not differ between trees grown in sun and shade conditions. Thus, differences in effects of predators on herbivores and plants between light habitats, when observed, might be due to variation in predator abundance and not bottom-up effects of host plant quality.     

Multivariate control of root biomass in a semi-arid grassland on the Loess Plateau,China

Aim

Root biomass has long been under-represented in biodiversity–ecosystem functioning studies, despite its dominance in biomass in many arid and semi-arid ecosystems. We aimed to explore the multivariate control over root biomass by plant diversity, together with other biotic and abiotic factors and to evaluate the relative importance of these factors.

Methods

Above- and below-ground traits of 13 communities and soil properties were measured in semi-arid grasslands on the Loess Plateau, China. Structural equation modeling (SEM) was used to evaluate the relative importance of the community and soil characteristics, emphasizing the direct and indirect effects of plant diversity on root biomass.

Results

Significant indirect effects of plant species richness on root biomass were found, although no direct correlation was detected between them. In the indirect pathways, plant species richness showed a positive effect on soil total nitrogen, but a significant negative influence on soil total carbon. Soil total nitrogen and plant diversity had the largest and smallest total effect respectively on root biomass in the model.

Conclusions

Plant species richness was not the strongest determinant of root biomass but had a significant indirect effect, mediated through soil total carbon and nitrogen. This study suggests that greater plant species richness, through a positive influence on soil total nitrogen, may indirectly promote root carbon stock.     

Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat.

White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.     

Mycorrhizal fungi as mediators of defence against insect pests in agricultural systems

• 1 Below‐ground organisms influence above‐ground interactions in both natural and agricultural ecosystems. Among the most important below‐ground organisms are mycorrhizal fungi, comprising ubiquitous and ancient plant mutualists that have significant effects on plant growth and fitness mediated by resource exchange with plants. In the present study, we focus on the effects of arbuscular mycorrhizal fungi (AMF) on crop defence against insect pests.
• 2 AMF alter the availability of resources used by crop plants to manufacture defences against pests and to compensate for pest damage. However, AMF also provide plants with nutrients that are known to increase insect performance. Through potentially opposing effects on plant nutritional quality and defence, mycorrhizal fungi can positively or negatively affect pest performance.
• 3 Additionally, AMF may directly affect gene expression and plant defence signalling pathways involved in the construction and induction of plant defences, and these effects are apparently independent of those caused by nutrient availability. In this way, AMF may still influence plant defences in the fertilized and highly managed systems typical of agribusiness.
• 4 Because AMF can affect plant tolerance to pest damage, they may have a significant impact on the shape of damage–yield relationships in crops. Potential mechanisms for this effect are suggested.
• 5 We highlight the need for continuing research on the effects of AMF identity and the abundance on crop defences and tolerance to pest attack. Much work is needed on the potential effects of mycorrhizal colonization on plant signalling and the induction of direct and indirect defences that may protect against pest damage.

     

Environmental heterogeneity mediates a cross‐ecosystem trophic cascade

1. The flow of energy and nutrients across ecosystem boundaries can have significant community‐wide effects, but the role of productivity of the recipient habitat in mediating these effects remains unclear. This is especially true when organisms moving across ecosystem boundaries serve simultaneously as predators and prey. 2. In this study, the effects of odonates, primarily Enallagma civile (Hagen), on a salt marsh system were examined. Cages were used to exclude odonate predators, but not other arthropods, from experimental plots of the sea oxeye daisy, Borrichia frutescens (L.) in high and low productivity areas. The effects were assessed on the in situ arthropod community and the host plant. 3. There were strong direct effects of predation on the herbivores Pissonotus quadripustulatus Van Duzee and Asphondylia borrichiae Rossi and Strong, with higher densities where damselflies were excluded. Damselflies also served as prey for web‐building spiders. This resulted in lower spider densities inside cages, and a positive indirect effect on grasshopper densities. 4. Direct and indirect effects of odonates were greater in the high productivity area, resulting in a trophic cascade, with greater damage and reduced flowering and density of the host plant inside cages. 5. The results of this study support the subsidy hypothesis and show that theoretical models of trophic dynamics, which were developed to explain exchanges within ecosystems, may have predictive and explanatory value for exchanges across ecosystems as well.     

Microgravity environment uncouples cell growth and cell proliferation in root meristematic cells: The mediator role of auxin

Experiments performed in space have evidenced that, in root meristematic cells, the absence of gravity results in the uncoupling of cell growth and cell proliferation, two essential cellular functions that support plant growth and development, which are strictly coordinated under normal ground gravity conditions. In space, cell proliferation appears enhanced whereas cell growth is depleted. Since coordination of cell growth and proliferation is a major feature of meristematic cells, the observed uncoupling is a serious stress condition for these cells producing important alterations in the developmental pattern of the plant. Auxin plays a major role in these processes both by assuring the coupling of cell growth and proliferation under normal conditions and by exerting a decisive influence in the uncoupling under altered gravity conditions. Auxin is a mediator of the transduction of the gravitropic signal and its distribution in the root is altered subsequent to a change in the gravity conditions. This altered distribution may produce changes in the expression of specific growth coordinators leading to the alteration of cell cycle and protein synthesis. Therefore, available data indicate that the effects of altered gravity on cell growth and proliferation are the consequence of the transduction of the gravitropic signal perceived by columella cells, in the root tip.Key words: cell cycle, ribosome biogenesis, nucleolus, auxin efflux, graviperception, space flight, arabidopsisThe size and morphology of plants and of plant organs is basically determined by cellular activities that occur in meristems. The primary meristems are root and shoot apical meristems, located at both upper and lower ends of the plant, which are constituted by stem cells. Cell division in these meristems is required to supply new cells for expansion and differentiation of tissues and initiation of new organs, providing the basic structure of the plant body.¹ In turn, active protein synthesis is required after mitosis in order to promote the necessary cell growth, up to duplication of cell size, which will make possible a new cell division. This continuous activity of growth and proliferation in meristematic cells is controlled by auxin, whose distribution in roots sets up distinct zones for cell division, cell expansion and differentiation and determines the balance between them.^2,3Therefore, cell growth and proliferation are essential functions for plant development and they are involved in the developmental response to environmental stimuli, such as tropisms and defense mechanisms against both biotic and abiotic agents.^4–6 Gravity is a fundamental environmental condition, constant in the Earth as a factor conditioning life throughout its whole history. Plants are particularly affected by gravity in their growth, which is directed by the gravity vector producing the well known process of gravitropism.An experiment aimed to know the effects of a weightless environment on cell proliferation and growth in root meristematic cells was performed in the International Space Station. It consisted of germinating seeds of Arabidopsis thaliana in space and then growing seedlings for four days at the constant temperature of 22°C, in the darkness. Seedlings were fixed when still in space and recovered on ground to be processed for microscopical study. In addition, samples from a previous space experiment, grown in a similar way but fixed differently and including a control flight experiment in a 1 g centrifuge, were also incorporated to the analysis.^7,8 This analysis consisted of biometrical estimations of the seedling and root length, quantitative measurements at the cellular level, including number of cells per millimeter in specific cell files, in order to get an estimate of the cell proliferation rate, and morphometrical, ultrastructural and immunocytochemical study of the nucleolus, in order to know the rate of ribosome biogenesis, as an estimation of the level of protein synthesis, which is the cellular process which determines cell growth in the root meristem. Data obtained from space-flown samples were compared with 1 g ground controls and also with data from samples grown in the same conditions in a device called “Random Positioning Machine”, an efficient simulator of microgravity, which induces constant changes of the gravity vector as it is sensed by living samples.⁹ The results interestingly showed an enhanced rate of cell proliferation accompanied by a reduction of ribosome biogenesis per cell in samples grown in both real and simulated microgravity, compared to 1 g controls, either in flight or on ground.¹⁰ This alteration of essential cellular processes may go far beyond the mere change in specific physiological activities of a particular cell type, since, on the one hand, alteration of cell growth and proliferation in the root meristem may have consequences at the level of development and shaping of the whole plant; on the other hand, regulation of these cellular activities by auxin may put in connection these cellular alterations with the transduction cascade of the gravitropic signal perceived by columella cells in the root tip, which is altered when the environmental gravity conditions change and which finally results in the modification of the levels and distribution of auxin throughout the root.     

Joining forces: The interface of gravitropism and plastid protein import

In flowering plants, gravity perception appears to involve the sedimentation of starch-filled plastids, called amyloplasts, within specialized cells (the statocytes) of shoots (endodermal cells) and roots (columella cells). Unfortunately, how the physical information derived from amyloplast sedimentation is converted into a biochemical signal that promotes organ gravitropic curvature remains largely unknown. Recent results suggest an involvement of the Translocon of the Outer Envelope of (Chloro) plastids (TOC) in early phases of gravity signal transduction within the statocytes. This review summarizes our current knowledge of the molecular mechanisms that govern gravity signal transduction in flowering plants and summarizes models that attempt to explain the contribution of TOC proteins in this important behavioral plant growth response to its mechanical environment.Key words: gravitropism, root, amyloplast, TOC complex, TOC132, TOC75     

Escherichia coli metabolism in space.

Cultures of the bacterium Escherichia coli were grown in the orbiting Biocosmos 2044 satellite in order to evaluate the effects of the space environment--weightlessness and heavy particle radiation--on growth parameters and energy metabolism, which have previously been reported to be affected, and on induction of the SOS response, which reflects DNA damage to the cell. We found no differences between the flight samples and control ground cultures in the growth yield per gram of carbon, in mean cell mass (from which we deduce that the growth rate was unaltered) or in the level of expression of the SOS response. These observations indicate that free-growing bacterial cells do not expend significant energy fighting gravity and that cosmic radiation within a space capsule does not produce significant levels of DNA damage.     

Effects of amount and recurrence of leaf herbivory on the induction of direct and indirect defences in wild cotton

• The induction of defences in response to herbivory is a key mechanism of plant resistance. While a number of studies have investigated the time course and magnitude of plant induction in response to a single event of herbivory, few have looked at the effects of recurrent herbivory. Furthermore, studies measuring the effects of the total amount and recurrence of herbivory on both direct and indirect plant defences are lacking. To address this gap, here we asked whether insect leaf herbivory induced changes in the amount and concentration of extrafloral nectar (an indirect defence) and concentration of leaf phenolic compounds (a direct defence) in wild cotton (Gossypium hirsutum).
• We conducted a greenhouse experiment where we tested single event or recurrent herbivory effects on defence induction by applying mechanical leaf damage and caterpillar (Spodoptera frugiperda) regurgitant.
• Single events of 25% and 50% leaf damage did not significantly influence extrafloral nectar production or concentration. Extrafloral nectar traits did, however, increase significantly relative to controls when plants were exposed to recurrent herbivory (two episodes of 25% damage). In contrast, phenolic compounds increased significantly in response to single events of  leaf damage but not to recurrent damage. In addition, we found. that local induction of extrafloral nectar production was stronger than systemic induction, whereas the reverse pattern was observed for phenolics.
• Together, these results reveal seemingly inverse patterns of induction of direct and indirect defences in response to herbivory in wild cotton.