Brachypodium distachyon Seedlings Display Accession-Specific Morphological and Transcriptomic Responses to the Microgravity Environment of the International Space Station.

Plants have been recognized as key components of bioregenerative life support systems for space exploration, and many experiments have been carried out to evaluate their adaptability to spaceflight. Unfortunately, few of these experiments have involved monocot plants, which constitute most of the crops used on Earth as sources of food, feed, and fiber. To better understand the ability of monocot plants to adapt to spaceflight, we germinated and grew Brachypodium distachyon seedlings of the Bd21, Bd21-3, and Gaz8 accessions in a customized growth unit on the International Space Station, along with 1-g ground controls. At the end of a 4-day growth period, seedling organ's growth and morphologies were quantified, and root and shoot transcriptomic profiles were investigated using RNA-seq. The roots of all three accessions grew more slowly and displayed longer root hairs under microgravity conditions relative to ground control. On the other hand, the shoots of Bd21-3 and Gaz-8 grew at similar rates between conditions, whereas those of Bd21 grew more slowly under microgravity. The three Brachypodium accessions displayed dramatically different transcriptomic responses to microgravity relative to ground controls, with the largest numbers of differentially expressed genes (DEGs) found in Gaz8 (4527), followed by Bd21 (1353) and Bd21-3 (570). Only 47 and six DEGs were shared between accessions for shoots and roots, respectively, including DEGs encoding wall-associated proteins and photosynthesis-related DEGs. Furthermore, DEGs associated with the "Oxidative Stress Response" GO group were up-regulated in the shoots and down-regulated in the roots of Bd21 and Gaz8, indicating that Brachypodium roots and shoots deploy distinct biological strategies to adapt to the microgravity environment. A comparative analysis of the Brachypodium oxidative-stress response DEGs with the Arabidopsis ROS wheel suggests a connection between retrograde signaling, light response, and decreased expression of photosynthesis-related genes in microgravity-exposed shoots. In Gaz8, DEGs were also found to preferentially associate with the "Plant Hormonal Signaling" and "MAP Kinase Signaling" KEGG pathways. Overall, these data indicate that Brachypodium distachyon seedlings exposed to the microgravity environment of ISS display accession- and organ-specific responses that involve oxidative stress response, wall remodeling, photosynthesis inhibition, expression regulation, ribosome biogenesis, and post-translational modifications. The general characteristics of these responses are similar to those displayed by microgravity-exposed Arabidopsis thaliana seedlings. However, organ- and accession-specific components of the response dramatically differ both within and between species. These results suggest a need to directly evaluate candidate-crop responses to microgravity to better understand their specific adaptability to this novel environment and develop cultivation strategies allowing them to strive during spaceflight.

Low-Speed Clinorotation of Brachypodium distachyon and Arabidopsis thaliana Seedlings Triggers Root Tip Curvatures That Are Reminiscent of Gravitropism.

Clinostats are instruments that continuously rotate biological specimens along an axis, thereby averaging their orientation relative to gravity over time. Our previous experiments indicated that low-speed clinorotation may itself trigger directional root tip curvature. In this project, we have investigated the root curvature response to low-speed clinorotation using Arabidopsis thaliana and Brachypodium distachyon seedlings as models. We show that low-speed clinorotation triggers root tip curvature in which direction is dictated by gravitropism during the first half-turn of clinorotation. We also show that the angle of root tip curvature is modulated by the speed of clinorotation. Arabidopsis mutations affecting gravity susception (pgm) or gravity signal transduction (arg1, toc132) are shown to affect the root tip curvature response to low-speed clinorotation. Furthermore, low-speed vertical clinorotation triggers relocalization of the PIN3 auxin efflux facilitator to the lateral membrane of Arabidopsis root cap statocytes, and creates a lateral gradient of auxin across the root tip. Together, these observations support a role for gravitropism in modulating root curvature responses to clinorotation. Interestingly, distinct Brachypodium distachyon accessions display different abilities to develop root tip curvature responses to low-speed vertical clinorotation, suggesting the possibility of using genome-wide association studies to further investigate this process.

Application of Cadaverine to Inhibit Biotin Biosynthesis in Plants.

Biotin is an essential vitamin in plants. However, characterization of biotin deficiency has been limited by embryo lethality in mutants, which can only be rescued by supplementation of biotin. Here, we describe a protocol to characterize biotin deficiency in Arabidopsis thaliana through application of the polyamine cadaverine. Cadaverine induces changes in primary root growth. Protein biotinylation in Arabidopsis seedlings can be quantified through an assay similar to a western blot, in which protein biotinylation is detected by a streptavidin probe. This technique provides a chemical means of inhibiting biotin synthesis, allowing for further characterization of biotin deficiency on a physiological and molecular level.

Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis.

Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.

Gravity Signaling in Flowering Plant Roots.

Roots typically grow downward into the soil where they anchor the plant and take up water and nutrients necessary for plant growth and development. While the primary roots usually grow vertically downward, laterals often follow a gravity set point angle that allows them to explore the surrounding environment. These responses can be modified by developmental and environmental cues. This review discusses the molecular mechanisms that govern root gravitropism in flowering plant roots. In this system, the primary site of gravity sensing within the root cap is physically separated from the site of curvature response at the elongation zone. Gravity sensing involves the sedimentation of starch-filled plastids (statoliths) within the columella cells of the root cap (the statocytes), which triggers a relocalization of plasma membrane-associated PIN auxin efflux facilitators to the lower side of the cell. This process is associated with the recruitment of RLD regulators of vesicular trafficking to the lower membrane by LAZY proteins. PIN relocalization leads to the formation of a lateral gradient of auxin across the root cap. Upon transmission to the elongation zone, this auxin gradient triggers a downward curvature. We review the molecular mechanisms that control this process in primary roots and discuss recent insights into the regulation of oblique growth in lateral roots and its impact on root-system architecture, soil exploration and plant adaptation to stressful environments.

Quantitative Trait Loci for Root Growth Response to Cadaverine in Arabidopsis.

Root growth architecture is a major determinant of agricultural productivity and plant fitness in natural ecosystems. Here we describe the methods used in a Quantitative Trait Loci (QTL) study that allowed the identification of ORGANIC CATION TRANSPORTER 1 (OCT1) as a determinant of root growth response to cadaverine treatment in Arabidopsis thaliana. This protocol screens natural accessions to characterize the variation in root growth response to the naturally occurring polyamine cadaverine, then uses recombination mapping to identify loci that are responsible for the variation existing between two accessions with contrasting phenotypes.

Molecular Mechanisms of Root Gravitropism.

Plant shoots typically grow against the gravity vector to access light, whereas roots grow downward into the soil to take up water and nutrients. These gravitropic responses can be altered by developmental and environmental cues. In this review, we discuss the molecular mechanisms that govern the gravitropism of angiosperm roots, where a physical separation between sites for gravity sensing and curvature response has facilitated discovery. Gravity sensing takes place in the columella cells of the root cap, where sedimentation of starch-filled plastids (amyloplasts) triggers a pathway that results in a relocalization to the lower side of the cell of PIN proteins, which facilitate efflux of the plant hormone auxin efflux. Consequently, auxin accumulates in the lower half of the root, triggering bending of the root tip at the elongation zone. We review our understanding of the molecular mechanisms that control this process in primary roots, and discuss recent insights into the regulation of oblique growth in lateral roots and its impact on root-system architecture and soil exploration.

Cadaverine's Functional Role in Plant Development and Environmental Response.

Cadaverine derives from lysine in a pathway that is distinct from that of the other well-characterized ornithine- or arginine-derived polyamines. Despite a multitude of studies in bacterial systems, cadaverine has garnered little attention in plant research. Nonetheless, many plants have been found to synthesize it. For instance, the Leguminosae have been shown to produce cadaverine and use it as a precursor in the biosynthesis of quinolizidine alkaloids, secondary metabolites that are involved in insect defense and also display therapeutic pharmacological properties. Cadaverine is also present in the environment; it can be produced by rhizosphere and phyllosphere microbes. Markedly, exogenous cadaverine application causes alterations in root-system architecture. Previous research suggests cadaverine has a role in stress response, with groups reporting an increase in content upon exposure to heat, drought, salt, and oxidative stress. However, data regarding the role of cadaverine in stress response remains conflicted, as some plant systems show enhanced tolerance to stresses in its presence, while others show increased sensitivity to the same stresses. In this review, we summarize recent findings on the role of cadaverine in plant growth, development, and stress response. We also address the possible roles rhizosphere and phyllosphere microbes may play in the delivery of exogenous cadaverine near plant organs, and discuss our current understanding of the molecular pathways that contribute to cadaverine homeostasis and response in plants.

Immunohistochemistry relative to gravity: a simple method to retain information about gravity for immunolocalization and histochemistry.

We describe a simple method to preserve information about a plant organ's orientation relative to the direction of the gravity vector during sample processing for immunolocalization or histochemical analysis of cell biological processes. This approach has been used in gravity stimulated roots of Arabidopsis thaliana and Zea mays to study PIN3 relocalization, study the asymmetrical remodeling of the actin network and the cortical microtubule array, and to reveal the asymmetrical expression of the auxin signaling reporter DR5::GUS. This method enables the rapid analysis of a large number of samples from a variety of genotypes, as well as from tissue that may be too thick for microscopy in live plants.

Natural variation in the expression of ORGANIC CATION TRANSPORTER 1 affects root length responses to cadaverine in Arabidopsis.

Polyamines, including cadaverine, are organic cations that affect numerous biological processes including transcription, translation, cell signalling, and ion channel activity. They often function in biotic and abiotic stress responses in plants. Because little is known about how plants respond to cadaverine, a quantitative natural variation approach was used to identify genetic factors that contribute to this response. Here it is shown that Arabidopsis thaliana accessions have varying root length responses to exogenous cadaverine: Cape Verde Islands (Cvi) was one of the most resistant accessions tested, whereas Landsberg erecta (Ler) was one of the most sensitive. Recombinant inbred lines, near isogenic lines, and a microarray were used to show that variation in ORGANIC CATION TRANSPORTER 1 (OCT1) is at least partially responsible for this difference. OCT1 expression was higher in Cvi than in Ler, and oct1 mutants were more sensitive to cadaverine than wild-type plants. In oct1 mutants transformed with an ectopic copy of OCT1 originating from either Cvi or Ler, the expression level of the transgene, not its accession, correlated with the cadaverine response. These results suggest that decreased OCT1 expression confers cadaverine sensitivity in some accessions.

The VASCULATURE COMPLEXITY AND CONNECTIVITY gene encodes a plant-specific protein required for embryo provasculature development.

The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins. Similar disconnections or gaps were observed in the provasculature of vcc embryos, indicating that defects in vein connectivity appear early in mutant embryo development. Consistently, the overexpression of VCC leads to an unusually high proportion of cotyledons with high-complexity vein networks. Neither auxin distribution nor the polar localization of the auxin efflux carrier were affected in vcc mutant embryos. Expression of VCC was detected in developing embryos and procambial, cambial, and vascular cells of cotyledons, leaves, roots, hypocotyls, and anthers. To evaluate possible genetic interactions with other genes that control vasculature patterning in embryos, we generated a double mutant for VCC and OCTOPUS (OPS). The vcc ops double mutant embryos showed a complete loss of high-complexity vascular networks in cotyledons and a drastic increase in both provascular and vascular disconnections. In addition, VCC and OPS interact physically, suggesting that VCC and OPS are part of a complex that controls cotyledon vascular complexity.

A functional TOC complex contributes to gravity signal transduction in Arabidopsis.

Although plastid sedimentation has long been recognized as important for a plant's perception of gravity, it was recently shown that plastids play an additional function in gravitropism. The Translocon at the Outer envelope membrane of Chloroplasts (TOC) complex transports nuclear-encoded proteins into plastids, and a receptor of this complex, Toc132, was previously hypothesized to contribute to gravitropism either by directly functioning as a gravity signal transducer or by indirectly mediating the plastid localization of a gravity signal transducer. Here we show that mutations in multiple genes encoding TOC complex components affect gravitropism in a genetically sensitized background and that the cytoplasmic acidic domain of Toc132 is not required for its involvement in this process. Furthermore, mutations in TOC132 enhance the gravitropic defect of a mutant whose amyloplasts lack starch. Finally, we show that the levels of several nuclear-encoded root proteins are altered in toc132 mutants. These data suggest that the TOC complex indirectly mediates gravity signal transduction in Arabidopsis and support the idea that plastids are involved in gravitropism not only through their ability to sediment but also as part of the signal transduction mechanism.

CACTIN is an essential nuclear protein in Arabidopsis and may be associated with the eukaryotic spliceosome.

CACTIN is a conserved eukaryotic protein without known functional domains. Previous research revealed that CACTIN is essential in animals and protists and that it may function in inflammation pathways in animals; however, these pathways are not as broadly conserved as CACTIN. Therefore, the ancestral molecular function of CACTIN remains unknown. Our studies using Arabidopsis show that CACTIN is required for embryogenesis. Fluorescently tagged CACTIN localizes to nuclear speckles and colocalizes with known splicing proteins. In yeast-two-hybrid studies, we found that CACTIN binds to a putative component of the spliceosome. These findings support a possible role for CACTIN in splicing.

Gravity sensing and signal transduction in vascular plant primary roots.

During gravitropism, the potential energy of gravity is converted into a biochemical signal. How this transfer occurs remains one of the most exciting mysteries in plant cell biology. New experiments are filling in pieces of the puzzle. In this review, we introduce gravitropism and give an overview of what we know about gravity sensing in roots of vascular plants, with special highlight on recent papers. When plant roots are reoriented sideways, amyloplast resedimentation in the columella cells is a key initial step in gravity sensing. This process somehow leads to cytoplasmic alkalinization of these cells followed by relocalization of auxin efflux carriers (PINs). This changes auxin flow throughout the root, generating a lateral gradient of auxin across the cap that upon transmission to the elongation zone leads to differential cell elongation and gravibending. We will present the evidence for and against the following players having a role in transferring the signal from the amyloplast sedimentation into the auxin signaling cascade: mechanosensitive ion channels, actin, calcium ions, inositol trisphosphate, receptors/ligands, ARG1/ARL2, spermine, and the TOC complex. We also outline auxin transport and signaling during gravitropism.

Multiple roles for membrane-associated protein trafficking and signaling in gravitropism.

Gravitropism is a process that allows plant organs to guide their growth relative to the gravity vector. It requires them to sense changes in their orientation and generate a biochemical signal that they transmit to the tissues that drive organ curvature. Trafficking between the plasma membrane and endosomal compartments is important for all of these phases of the gravitropic response. The sedimentation of starch-filled organelles called amyloplasts plays a key role in sensing reorientation, and vacuolar integrity is required for amyloplast sedimentation in shoots. Other proteins associated with the vesicle trafficking pathway contribute to early gravity signal transduction independently of amyloplast sedimentation in both roots and hypocotyls. Phosphatidylinositol signaling, which starts at the plasma membrane and later affects the localization of auxin efflux facilitators, is a likely second messenger in the signal transduction phase of gravitropism. Finally, membrane-localized auxin influx and efflux facilitators contribute to a differential auxin gradient across the gravistimulated organs, which directs root curvature.

Molecular mechanisms of root gravity sensing and signal transduction.

Plants use gravity as a guide to direct their roots down into the soil to anchor themselves and to find resources needed for growth and development. In higher plants, the columella cells of the root tip form the primary site of gravity sensing, and in these cells the sedimentation of dense, starch-filled plastids (amyloplasts) triggers gravity signal transduction. This generates an auxin gradient across the root cap that is transmitted to the elongation zone where it promotes differential cell elongation, allowing the root to direct itself downward. It is still not well understood how amyloplast sedimentation leads to auxin redistribution. Models have been proposed to explain how mechanosensitive ion channels or ligand-receptor interactions could connect these events. Although their roles are still unclear, possible second messengers in this process include protons, Ca(2+), and inositol 1,4,5-triphosphate. Upon gravistimulation, the auxin efflux facilitators PIN3 and PIN7 relocalize to the lower side of the columella cells and mediate auxin redistribution. However, evidence for an auxin-independent secondary mechanism of gravity sensing and signal transduction suggests that this physiological process is quite complex. Furthermore, plants must integrate a variety of environmental cues, resulting in multifaceted relationships between gravitropism and other directional growth responses such as hydro-, photo-, and thigmotropism.

Studying starch content and sedimentation of amyloplast statoliths in Arabidopsis roots.

Amyloplasts, organelles responsible for the synthesis and storage of starch, are of critical importance to gravitropism in higher plants. We discuss two methods that are useful for describing the histology and behavior of amyloplasts. First, because mutants with little or no plastidic starch accumulation are defective in their gravitropic response, we review a method to observe starch accumulation quickly in plant tissue. Second, we discuss a method for measuring amyloplast sedimentation in the dynamic environment of Arabidopsis root columella cells, which is thought to provide a directional cue to a reoriented plant.