Fundamental Biological Features of Spaceflight: Advancing the Field to Enable Deep-Space Exploration.

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

The vacuolar Ca2+ transporter CATION EXCHANGER 2 regulates cytosolic calcium homeostasis, hypoxic signaling, and response to flooding in Arabidopsis thaliana.

Flooding represents a major threat to global agricultural productivity and food security, but plants are capable of deploying a suite of adaptive responses that can lead to short- or longer-term survival to this stress. One cellular pathway thought to help coordinate these responses is via flooding-triggered Ca2+ signaling. We have mined publicly available transcriptomic data from Arabidopsis subjected to flooding or low oxygen stress to identify rapidly upregulated, Ca2+ -related transcripts. We then focused on transporters likely to modulate Ca2+ signals. Candidates emerging from this analysis included AUTOINHIBITED Ca2+ ATPASE 1 and CATION EXCHANGER 2. We therefore assayed mutants in these genes for flooding sensitivity at levels from growth to patterns of gene expression and the kinetics of flooding-related Ca2+ changes. Knockout mutants in CAX2 especially showed enhanced survival to soil waterlogging coupled with suppressed induction of many marker genes for hypoxic response and constitutive activation of others. CAX2 mutants also generated larger and more sustained Ca2+ signals in response to both flooding and hypoxic challenges. CAX2 is a Ca2+ transporter located on the tonoplast, and so these results are consistent with an important role for vacuolar Ca2+ transport in the signaling systems that trigger flooding response.

The vacuolar H+/Ca transporter CAX1 participates in submergence and anoxia stress responses.

A plant's oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice (Oryza sativa) cultivation, and submergence tolerance of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters to this process is poorly understood. Four functional cation/proton exchangers (CAX1-4) in Arabidopsis (Arabidopsis thaliana) help regulate Ca homeostasis around the vacuole. Our results demonstrate that cax1 mutants are more tolerant to both anoxic conditions and submergence. Using phenotypic measurements, RNA-sequencing, and proteomic approaches, we identified cax1-mediated anoxia changes that phenocopy changes present in anoxia-tolerant crops: altered metabolic processes, diminished reactive oxygen species production post anoxia, and altered hormone signaling. Comparing wild-type and cax1 expressing genetically encoded Ca indicators demonstrated altered cytosolic Ca signals in cax1 during reoxygenation. Anoxia-induced Ca signals around the plant vacuole are involved in the control of numerous signaling events related to adaptation to low oxygen stress. This work suggests that cax1 anoxia response pathway could be engineered to circumvent the adverse effects of flooding that impair production agriculture.

Tonoplast-localized Ca2+ pumps regulate Ca2+ signals during pattern-triggered immunity in Arabidopsis thaliana.

One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.

CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling.

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca2+-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca2+ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca2+ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca2+ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS-YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca2+ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding.

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction.

Changes in Nuclear Shape and Gene Expression in Response to Simulated Microgravity Are LINC Complex-Dependent.

Microgravity is known to affect the organization of the cytoskeleton, cell and nuclear morphology and to elicit differential expression of genes associated with the cytoskeleton, focal adhesions and the extracellular matrix. Although the nucleus is mechanically connected to the cytoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, the role of this group of proteins in these responses to microgravity has yet to be defined. In our study, we used a simulated microgravity device, a 3-D clinostat (Gravite), to investigate whether the LINC complex mediates cellular responses to the simulated microgravity environment. We show that nuclear shape and differential gene expression are both responsive to simulated microgravity in a LINC-dependent manner and that this response changes with the duration of exposure to simulated microgravity. These LINC-dependent genes likely represent elements normally regulated by the mechanical forces imposed by gravity on Earth.

Variation in the transcriptome of different ecotypes of Arabidopsis thaliana reveals signatures of oxidative stress in plant responses to spaceflight.

PREMISE OF THE STUDY: Spaceflight provides a unique environment in which to dissect plant stress response behaviors and to reveal potentially novel pathways triggered in space. We therefore analyzed the transcriptomes of Arabidopsis thaliana plants grown on board the International Space Station to find the molecular fingerprints of these space-related response networks. METHODS: Four ecotypes (Col-0, Ws-2, Ler-0 and Cvi-0) were grown on orbit and then their patterns of transcript abundance compared to ground-based controls using RNA sequencing. KEY RESULTS: Transcripts from heat-shock proteins were upregulated in all ecotypes in spaceflight, whereas peroxidase transcripts were downregulated. Among the shared and ecotype-specific changes, gene classes related to oxidative stress and hypoxia were detected. These spaceflight transcriptional response signatures could be partly mimicked on Earth by a low oxygen environment and more fully by oxidative stress (H2 O2 ) treatments. CONCLUSIONS: These results suggest that the spaceflight environment is associated with oxidative stress potentially triggered, in part, by hypoxic response. Further, a shared spaceflight response may be through the induction of molecular chaperones (such as heat shock proteins) that help protect cellular machinery from the effects of oxidative damage. In addition, this research emphasizes the importance of considering the effects of natural variation when designing and interpreting changes associated with spaceflight experiments.

Quantitative ROS bioreporters: A robust toolkit for studying biological roles of ROS in response to abiotic and biotic stresses.

While the accumulation of reactive oxygen species (ROS) through spontaneous generation or as the by-products of aerobic metabolism can be toxic to plants, recent findings demonstrate that ROS act as signaling molecules that play a critical role in adapting to various stress conditions. Tight regulation of ROS homeostasis is required to adapt to stress and survive, yet in vivo spatiotemporal information of ROS dynamics are still largely undefined. In order to understand the dynamics of ROS changes and their biological function in adapting to stresses, two quantitative ROS transcription-based bioreporters were developed. These reporters use ROS-responsive promoters from RBOHD or ZAT12 to drive green fluorescent protein (GFP) expression. The resulting GFP expression is compared to a constitutively expressed mCherry that is contained on the same cassette with the ROS-responsive promoter: This allows for the generation of ratiometric images comparing ROS changes (GFP) to the constitutively expressed mCherry. Both reporters were used to assess ROS levels to oxidative stress, salt stress, and the pathogen defense elicitor flg22. These bioreporters showed increases in the ratio values of GFP to mCherry signals between 10 and 30 min poststress application. Such stress-associated ROS signals correlated with the induction of abiotic/biotic stress responsive markers such as RbohD, ZAT12, SOS2 and PR5 suggesting these ROS bioreporters provide a robust indicator of increased ROS related to stress responses. Based upon the spatiotemporal response patterns of signal increase, ZAT12 promoter-dependent ROS (Zat12p-ROS) bioreporter appears to be suitable for cellular mapping of ROS changes in response to abiotic and biotic stresses.

Orchestrating rapid long-distance signaling in plants with Ca2+ , ROS and electrical signals.

Plants show a rapid systemic response to a wide range of environmental stresses, where the signals from the site of stimulus perception are transmitted to distal organs to elicit plant-wide responses. A wide range of signaling molecules are trafficked through the plant, but a trio of potentially interacting messengers, reactive oxygen species (ROS), Ca2+ and electrical signaling ('trio signaling') appear to form a network supporting rapid signal transmission. The molecular components underlying this rapid communication are beginning to be identified, such as the ROS producing NAPDH oxidase RBOHD, the ion channel two pore channel 1 (TPC1), and glutamate receptor-like channels GLR3.3 and GLR3.6. The plant cell wall presents a plant-specific route for possible propagation of signals from cell to cell. However, the degree to which the cell wall limits information exchange between cells via transfer of small molecules through an extracellular route, or whether it provides an environment to facilitate transmission of regulators such as ROS or H+ remains to be determined. Similarly, the role of plasmodesmata as both conduits and gatekeepers for the propagation of rapid cell-to-cell signaling remains a key open question. Regardless of how signals move from cell to cell, they help prepare distant parts of the plant for impending challenges from specific biotic or abiotic stresses.

Plants eavesdrop on cues produced by snails and induce costly defenses that affect insect herbivores.

Although induced defenses are widespread in plants, the degree to which plants respond to herbivore kairomones (incidental chemicals that herbivores produce independent of herbivory), the costs and benefits of responding to cues of herbivory risk, and the ecological consequences of induced defenses remain unclear. We demonstrate that undamaged tomatoes, Solanum lycopersicum, induce defenses in response to a kairomone (locomotion mucus) of snail herbivores (Helix aspersa). Induced defense had significant costs and benefits for plants: plants exposed to snail mucus or a standard defense elicitor (methyl jasmonate, MeJA) exhibited slower growth, but also experienced less herbivory by an insect herbivore (Spodoptera exigua). We also find that kairomones from molluscan herbivores lead to deleterious effects on insect herbivores mediated through changes in plant defense, i.e., mucus-induced defenses of Solanum lycopersicum-reduced growth of S. exigua. These results suggest that incidental cues of widespread generalist herbivores might be a mechanism creating variation in plant growth, plant defense, and biotic interactions.

Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species.

Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli.

Control of basal jasmonate signalling and defence through modulation of intracellular cation flux capacity.

Unknown mechanisms tightly regulate the basal activity of the wound-inducible defence mediator jasmonate (JA) in undamaged tissues. However, the Arabidopsis fatty acid oxygenation upregulated2 (fou2) mutant in vacuolar two-pore channel 1 (TPC1D454N ) displays high JA pathway activity in undamaged leaves. This mutant was used to explore mechanisms controlling basal JA pathway regulation. fou2 was re-mutated to generate novel 'ouf' suppressor mutants. Patch-clamping was used to examine TPC1 cation channel characteristics in the ouf suppressor mutants and in fou2. Calcium (Ca2+ ) imaging was used to study the effects fou2 on cytosolic Ca2+ concentrations. Six intragenic ouf suppressors with near wild-type (WT) JA pathway activity were recovered and one mutant, ouf8, affected the channel pore. At low luminal calcium concentrations, ouf8 had little detectable effect on fou2. However, increased vacuolar Ca2+ concentrations caused channel occlusion, selectively blocking K+ fluxes towards the cytoplasm. Cytosolic Ca2+ concentrations in unwounded fou2 were found to be lower than in the unwounded WT, but they increased in a similar manner in both genotypes following wounding. Basal JA pathway activity can be controlled solely by manipulating endomembrane cation flux capacities. We suggest that changes in endomembrane potential affect JA pathway activity.

A ROS-Assisted Calcium Wave Dependent on the AtRBOHD NADPH Oxidase and TPC1 Cation Channel Propagates the Systemic Response to Salt Stress.

Plants exhibit rapid, systemic signaling systems that allow them to coordinate physiological and developmental responses throughout the plant body, even to highly localized and quickly changing environmental stresses. The propagation of these signals is thought to include processes ranging from electrical and hydraulic networks to waves of reactive oxygen species (ROS) and cytoplasmic Ca(2+) traveling throughout the plant. For the Ca(2+) wave system, the involvement of the vacuolar ion channel TWO PORE CHANNEL1 (TPC1) has been reported. However, the precise role of this channel and the mechanism of cell-to-cell propagation of the wave have remained largely undefined. Here, we use the fire-diffuse-fire model to analyze the behavior of a Ca(2+) wave originating from Ca(2+) release involving the TPC1 channel in Arabidopsis (Arabidopsis thaliana). We conclude that a Ca(2+) diffusion-dominated calcium-induced calcium-release mechanism is insufficient to explain the observed wave transmission speeds. The addition of a ROS-triggered element, however, is able to quantitatively reproduce the observed transmission characteristics. The treatment of roots with the ROS scavenger ascorbate and the NADPH oxidase inhibitor diphenyliodonium and analysis of Ca(2+) wave propagation in the Arabidopsis respiratory burst oxidase homolog D (AtrbohD) knockout background all led to reductions in Ca(2+) wave transmission speeds consistent with this model. Furthermore, imaging of extracellular ROS production revealed a systemic spread of ROS release that is dependent on both AtRBOHD and TPC1 These results suggest that, in the root, plant systemic signaling is supported by a ROS-assisted calcium-induced calcium-release mechanism intimately involving ROS production by AtRBOHD and Ca(2+) release dependent on the vacuolar channel TPC1.

Salt stress-induced Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in plants.

Their sessile lifestyle means that plants have to be exquisitely sensitive to their environment, integrating many signals to appropriate developmental and physiological responses. Stimuli ranging from wounding and pathogen attack to the distribution of water and nutrients in the soil are frequently presented in a localized manner but responses are often elicited throughout the plant. Such systemic signaling is thought to operate through the redistribution of a host of chemical regulators including peptides, RNAs, ions, metabolites, and hormones. However, there are hints of a much more rapid communication network that has been proposed to involve signals ranging from action and system potentials to reactive oxygen species. We now show that plants also possess a rapid stress signaling system based on Ca(2+) waves that propagate through the plant at rates of up to ∼ 400 µm/s. In the case of local salt stress to the Arabidopsis thaliana root, Ca(2+) wave propagation is channeled through the cortex and endodermal cell layers and this movement is dependent on the vacuolar ion channel TPC1. We also provide evidence that the Ca(2+) wave/TPC1 system likely elicits systemic molecular responses in target organs and may contribute to whole-plant stress tolerance. These results suggest that, although plants do not have a nervous system, they do possess a sensory network that uses ion fluxes moving through defined cell types to rapidly transmit information between distant sites within the organism.

The Rice E3-Ubiquitin Ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 Modulates the Expression of ROOT MEANDER CURLING, a Gene Involved in Root Mechanosensing, through the Interaction with Two ETHYLENE-RESPONSE FACTOR Transcription Factors.

Plant roots can sense and respond to a wide diversity of mechanical stimuli, including touch and gravity. However, little is known about the signal transduction pathways involved in mechanical stimuli responses in rice (Oryza sativa). This work shows that rice root responses to mechanical stimuli involve the E3-ubiquitin ligase rice HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (OsHOS1), which mediates protein degradation through the proteasome complex. The morphological analysis of the roots in transgenic RNA interference::OsHOS1 and wild-type plants, exposed to a mechanical barrier, revealed that the OsHOS1 silencing plants keep a straight root in contrast to wild-type plants that exhibit root curling. Moreover, it was observed that the absence of root curling in response to touch can be reverted by jasmonic acid. The straight root phenotype of the RNA interference::OsHOS1 plants was correlated with a higher expression rice ROOT MEANDER CURLING (OsRMC), which encodes a receptor-like kinase characterized as a negative regulator of rice root curling mediated by jasmonic acid. Using the yeast two-hybrid system and bimolecular fluorescence complementation assays, we showed that OsHOS1 interacts with two ETHYLENE-RESPONSE FACTOR transcription factors, rice ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN1 (OsEREBP1) and rice OsEREBP2, known to regulate OsRMC gene expression. In addition, we showed that OsHOS1 affects the stability of both transcription factors in a proteasome-dependent way, suggesting that this E3-ubiquitin ligase targets OsEREBP1 and OsEREBP2 for degradation. Our results highlight the function of the proteasome in rice response to mechanical stimuli and in the integration of these signals, through hormonal regulation, into plant growth and developmental programs.

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls.

Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 µm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception.

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope.

The starch-statolith hypothesis proposes that starch-filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so-called 'static' or 'settled' statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom-designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild-type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity-driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity-induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild-type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the 'bottom' of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.