Plants in Microgravity: Molecular and Technological Perspectives

Plants in Microgravity: Molecular and Technological Perspectives

Abstract: Plants are vital components of our ecosystem for a balanced life here on Earth, as a source of both food and oxygen for survival. Recent space exploration has extended the field of plant biology, allowing for future studies on life support farming on distant planets. This exploration will utilize life support technologies for long-term human space flights and settlements. Such longer space missions will depend on the supply of clean air, food, and proper waste management. The ubiquitous force of gravity is known to impact plant growth and development. Despite this, we still have limited knowledge about how plants can sense and adapt to microgravity in space. Thus, the ability of plants to survive in microgravity in space settings becomes an intriguing topic to be investigated in detail. The new knowledge could be applied to provide food for astronaut missions to space and could also teach us more about how plants can adapt to unique environments. Here, we briefly review and discuss the current knowledge about plant gravity-sensing mechanisms and the experimental possibilities to research microgravity-effects on plants either on the Earth or in orbit.

One. Introduction

Plants are an important element of life support systems in space exploration due to the fact that they provide essential components for the long-term extra-terrestrial survival of humans. They can be used in bio-regenerative life support systems as a source of food and oxygen, for the removal of carbon dioxide, and for the recycling of waste during space missions. As well as improving the atmosphere of closed environments, plants can also provide good psychological health benefits for astronauts. However, plants must be able to adapt to and grow in extra-terrestrial environments to provide the above functions.

Plants respond to environmental signals and changes by reorienting their growth. They adjust their orientation through the differential growth of their various regions. Such directed growth responses in plants are termed tropisms. Plant shoots generally grow against the gravity vector and towards light to support photosynthesis, whereas roots grow in the direction of gravity and away from light to acquire the water and nutrients from the soil. Unlike other changing biotic and abiotic conditions, gravity is the sole stable environmental factor to which plants had to adapt during their evolution. While land plants have evolved by adaptation to a gravitational force equal to one-G, the growth of aquatic species is adjusted to a lower value. The gravitropic signaling and response mechanism of plants grown on Earth has long been an area of interest to researchers. How these mechanisms influence plant growth-orientation under extra-terrestrial conditions is less understood. In an experiment, the gravi- and phototropic responses of Arabidopsis thaliana plants are investigated under red- and blue-light irradiation at microgravity settings of zero point one G, zero point three G, and one point zero G, modeling environmental conditions on the moon (zero point one seven G) or Mars (zero point three eight G), respectively. This study shows that directional light could act as a signal to govern directional plant growth even in the case of long-term low-gravity conditions. Nevertheless, further understanding of the molecular processes underlying gravity sensing could help to predict and/or manipulate plant behavior under microgravity conditions. Here we review the current knowledge on the root gravitropism in the model plant A. thaliana, investigate what is the best-studied system, and review the various experimental systems that can be used to investigate the effect of microgravity on plant behavior.

Two. Molecular Aspects of Plant Gravity Responses on Earth

Gravity, as a force, is the steadiest external stimulus on Earth that influences plant growth and development, and hence total plant performance. The typical reaction of plants to gravity is the growth of roots towards and the shoots against the gravity vector. Differential cell elongation induced by gravity and mediated by auxin in proximal and distal tissues results in the downward curvature of roots to acquire water and nutrients and the upward curvature of shoots to reach for sunlight. Primary roots and shoots usually tend to grow vertically, but lateral roots and shoot branches are oriented at a different angle to the gravity vector. This angle is denoted as the gravitropic set-point angle that is characteristic for plant species. The degree of the gravitropic set-point angle of lateral organs is set by environmental signals and developmentally controlled gravity-sensing and response mechanisms.

The molecular mechanism of plant gravity-sensing and response has been an important topic of debate in past decades raising several hypotheses. Gravity-sensing in plants was first explained by the still widely accepted starch (amyloplast)-statolith hypothesis. Statoliths are membrane-bound starch grains (amyloplasts) in gravity-detecting cells that can be found in the shoot cortex and the root columella. The basis of the theory is that statoliths sink to the bottom of reoriented gravity-sensing cells due to the change in the gravity vector. According to the hypothesis, the settling of amyloplasts is the primary component of gravity-sensing that triggers downstream signaling, generating biochemical and physiological responses in the responsive plant tissues. A less popular gravitational pressure model states that the whole protoplast, the entire cell content, has a role in sensing gravity. This model explains that mutants lacking starch in their plastids still respond to gravity, although with a reduced sensitivity. However, the importance of statoliths in gravity-sensing could not be questioned, as it was supported by several other experimental observations. A recent hypothesis, the graviproprioceptive drive theory, explains the limited but still important role of statoliths. Some models indicate that statolith movement and cell reorientation are in continuous feedback, resulting in oscillating growth. Based on this, the graviproprioceptive drive theory proposes that the straightness of the stem is attained by gravicline-sensing provided by statoliths together with the proprioception of tissue bending, self-sensing of tension and deformation.

The mechanism of organ bending in response to directional signals, tropism, was proposed by Cholodny and Went. According to their model, the gravitropic response, for example, relies on the lateral transport of auxin establishing unequal concentrations at the two sides of the organ, towards and away from the gravity signal, resulting in differential cell elongation and organ bending.

The role of auxin transport in gravitropism was underlined by applying the auxin transport inhibitor, NPA, preventing gravitropic root bending. Secondary metabolites such as flavonoids act as endogenous negative regulators of auxin transport. The mutation transparent testa that disrupts a gene that encodes for chalcone synthase, the first enzyme of flavonoid biosynthesis, leads to an increase in root growth and gravitropism due to an increment of auxin transport. The transparent testa mutants have substantially higher levels of transcripts of the PIN-FORMED protein two than the wild type, which aids in basipetal auxin transport in the root. Auxin and ethylene hormone interaction controls flavonol production via a signaling network involving TRANSPORT INHIBITOR RESPONSE ONE and ETHYLENE INSENSITIVE TWO/ETHYLENE RESISTANT ONE, which both converge on the MYB twelve transcription factor.

The gravity-induced asymmetrical auxin distribution at the root tips is established and maintained by various auxin influx and efflux transporters, including the AUX one/LAX (AUXIN-RESISTANT MUTATION one/LIKE AUX one) proteins, the ATP BINDING CASSETTE B/MULTIDRUG-RESISTANCE/P-GLYCOPROTEINS, and the PIN-FORMED proteins. The polar distribution of auxin is primarily governed by the PIN auxin efflux carriers, which are polarly localized in the cell membrane, pumping the auxin only in a defined direction. The PIN one, two, three, four, and seven proteins collectively control auxin distribution in the primary root. These proteins are present in specific, but overlapping, root tip regions. The PIN one mainly resides in the basal membrane of vascular parenchyma cells, and contributes to the accumulation of shoot-derived auxin in the root meristem. An incorrect expression or localization of PIN one in hsp ninety mutant roots prevented the gravitropic response. The PIN two is localized apically in the PM membranes of epidermal and lateral root cap cells and basally in cortical cells. Thus, PIN two transports auxin towards the shoot in epidermal cells but towards the tip in cortical cells, generating an auxin transport loop that was found to be important for the gravitropic response. The PIN two mutants (designated as eir one/agr one/pin two/wav six) exhibited an impaired root gravity response. The PIN three and PIN seven are expressed in the lateral root cap columella cells without pronounced polarity, and are also present in specific regions of the meristem. FOUR LIPS controls PIN three and PIN seven transcription levels in A. thaliana. Consequently, the auxin level was lower in the gravity-sensing cells of the flp mutant compared with wild-type roots. The PIN four can be observed in cells surrounding the quiescent centre and in the basal membrane of provascular cells. While pin two mutants exhibited an agravitropic phenotype, the pin three, pin four, and pin seven mutants were strongly gravitropic. Nevertheless, all these members of the PIN family have been related to the root response to gravity, probably with partly redundant roles. According to the current model, upon change in the orientation of the root, the PIN three and PIN seven transporters relocalize towards the lower side of the plasma membrane, directing auxin flow into the lower tiers of lateral root cap cells. Once asymmetry in auxin accumulation is initiated by PIN three and PIN seven, the PIN two transporter asymmetrically accumulates in the upper and lower sides of the root. As a consequence, auxin is transported in higher quantities in the lower epidermal cell tiers by PIN two, thus inhibiting cell elongation that results in the downward turning of the growing root.

The polar localization of PIN proteins at specific membrane domains is a dynamic process due to their endocytic vesicular recycling. The PIN polarity is controlled by posttranslational modifications (phosphorylation and ubiquitination). PINs are phosphorylated at specific residues by specific kinases. These kinases include the PINIOD, the WAG two, and the CDPK-RELATED KINASE five. Both loss-of-function and gain-of-function mutants of PINIOD exhibit defective PIN three polarization, and thus prevents bending in response to gravity. The PIN three phosphorylation is, therefore, essential for the gravitropic response. Furthermore, the PIN two-dependent basipetal auxin transport is also reduced in piniod-nine mutant roots. The CDPK-RELATED KINASE five was shown to be able to phosphorylate the hydrophilic loop of PIN two, and the Atcrk five mutant also exhibited a delayed root gravitropic response, correlated with inhibited redistribution of PIN two and limited auxin accumulation in the root tip region. The CDPK-RELATED KINASE five kinase can also phosphorylate the hydrophilic loop of other PIN proteins, including PIN three. Recently, AtCDPK-RELATED KINASE five was shown to have a role in maintaining the balance between reactive oxygen species and nitric oxide during root gravitropism in A. thaliana. The asymmetric redistribution of both reactive oxygen species and nitric oxide production is induced by auxin during the gravitropic response of roots, and both were shown to affect PIN two turnover and consequently auxin transport. Based on these observations, a regulatory feedback loop involving auxin, reactive oxygen species, and nitric oxide operating during the early gravitropic response of roots was hypothesized.

The role of cytoskeleton in plant gravitropism attracts attention from time to time. According to the tensegrity model of gravitropism, the plant cytoskeleton acts as a possible sensor and transmitter of the gravitropic signal. Microtubules, filamentous actin, and numerous regulatory proteins make up the cytoskeleton system. Using microtubule or actin polymerisation inhibitors in plants has highlighted the importance of the cytoskeleton in gravitropism. However, the significance of actin in gravitropism is debated, since disruption of the actin network did not always result in an agravitropic phenotype but could even enhance statolith sedimentation and the gravitropic response. The actin cytoskeleton is therefore believed to not be crucial for gravity-sensing per se, but for controlling the resting and sedimentation of statoliths. In agreement, the plastid-localized SHOOT GRAVITROPISM RESPONSE nine C three H two C three ring finger protein with ubiquitin E three ligase activity was shown to be required for amyloplast dissociation from the actin filaments. The sgr nine mutant exhibits reduced gravitropism since its amyloplasts cannot sediment. In contrast, in the fiz one Arabidopsis line having fragmented actin filaments due to a mutant ACTIN eight gene, the statoliths settle almost without hindrance. The ubiquitin ligase activity of SGR nine was found to be necessary for gravity-sensing, involving the protein degradation mechanism in the actin-mediated regulation of statolith dynamics. This view is strengthened by the fact that another E three ubiquitin ligase, WAVY GROWTH three, was also shown to be required for the root gravitropic response. The homologs of WAVY GROWTH three, including EDA forty, WAVY GROWTH H one, and WAVY GROWTH H two, have redundant positive functions in gravitropism, and the wav three wav H one wav H two triple mutant revealed auxin-signalling defects during the gravitropic response in roots. The identification of the protein targets of E three ligases implicated in gravitropism could highlight important details linking statolith dynamics to auxin distribution. Recent discoveries led to the hypothesis that members of the LAZY one-LIKE family of proteins have an important role in this link. The phenotypic characterization of four members of the Arabidopsis thaliana LAZY gene family revealed that the four members expressed in statocytes in both the shoot and the root contribute to gravitropism, as well as to the setting of the GSA. The defect in the gravitropic bending of lzy multiple mutants was due to their impairment in lateral auxin redistribution without the impact on amyloplast sedimentation on realignment.

Arabidopsis thaliana lateral roots emerge perpendicular to the primary root, but soon attain a GSA of approximately seventy-five degrees. This is due to the interaction of gravitropic and antigravitropic offset growth components, which are both auxin transport-dependent. The results suggest that GSAs are maintained by the interaction of the two opposing auxin fluxes, the balance of which determines the angle of organ growth. In agreement, following their emergence, lateral roots maintain a gravity-dependent non-vertical growth that progressively turns to vertical over several days. During this growth period, the lateral roots can reorientate their growth either upwards or downwards to maintain their GSA. In the lateral root tips of lzy mutants, the asymmetric distribution of PIN three and auxin response were reversed, suggesting that LZYs control asymmetric PIN three accumulation and the direction of polar auxin transport in the root cap columella in response to gravity. An analysis of the GSAs of the lateral roots of pin three-four, pin four-three, and pin seven-two single and multiple mutants led to the hypothesis that the repression of PIN four and PIN seven in the gravity-sensing columella cells is the cause of the limited gravitropic response of young lateral roots, in comparison with the main root where all three PINs are expressed in the statocytes. This is believed to strengthen the gravitropic response, allowing for the predominantly vertical growth of the main root. Recently, the LZY-interactor RCC one-like domain proteins were also reported as essential regulators of polar auxin transport and GSA. The following model emerged: statolith sedimentation first results in the polarization of the cellular localization of LZY that recruits RCC one-like domain one, which in turn is required for the relocalization of PIN three modulating the auxin flow. The transmembrane protein, ALTERED RESPONSE TO GRAVITY one, and its paralog ALTERED RESPONSE TO GRAVITY one-LIKE two were also implicated in PIN three relocalization and asymmetrical auxin distribution during gravistimulation. Both altered response to gravity one and altered response to gravity one-LIKE two mutations exhibit slower reorientation of hypocotyls and roots, without affecting the root starch content. It was hypothesized that these membrane-associated DnaJ proteins might have a role in the cellular polarization of LZY. The current molecular model of sensing gravity in roots is schematically summarized in Figure two.