Method for maintaining auxin flux in plants under microgravity conditions using leucoplastic mRNA (AEM2)

Abstract

The use of exogenous mRNA isolated from leucoplasts of germinating plants (AEM2-mRNA) for enhancing directed auxin transport under conditions of reduced gravity is described, wherein the mRNA comprises sequences encoding proteins of the PIN and AUX1 / LAX transport systems or their regulatory kinases, thereby stabilizing intrinsic cellular polarity and maintaining directed auxin translocation.

Description

[0001] Method for maintaining auxin flux in plants under microgravity by applying leucoplast mRNA (AEM2)

[0002] Background of the invention

[0003] In terrestrial environments, plant root cells use statoliths to sense gravitational signals and direct auxin transport for organized root and shoot growth. In microgravity, statoliths lose their ability to guide root growth, resulting in disrupted auxin gradients and irregular growth patterns. This lack of directional cues not only impairs root orientation but also disrupts several essential growth functions, such as lateral root formation, root meristem activity, and nutrient uptake.

[0004] In addition to the challenges of weightlessness, similar impairments of auxin transport also occur under terrestrial stress conditions, such as drought, heat, or nutrient deficiency. These factors also lead to a disruption of cellular polarity and can trigger growth arrest or dormancy.

[0005] Conventional approaches to address these deficiencies in space-based plant breeding have included genetic modification and hormone applications. However, these methods cannot fully compensate for the lack of gravity-dependent stimuli. This invention presents a novel solution: the application of AEM2 mRNA (auxin enhancement and morphogenesis mRNA), which optimizes the intrinsic polarity of cells and the function of auxin transport proteins, particularly PIN and AUX1 / LAX, to establish internal auxin gradients and stabilize the direction of root growth in microgravity.

[0006] Detailed description of the invention

[0007] The leucoplast mRNAs used in the present invention exhibit high compatibility with the cytoplasmic translation systems of the target plant cells. This compatibility is based on the optimized codon utilization of the leucoplast transcripts, which is evolutionarily adapted to the tRNA availability of germinating plant cells.

[0008] Leucoplasts, like chloroplasts, are of endosymbiotic origin and derive from photosynthetic cyanobacteria. Their gene expression has adapted over the course of evolution such that a large proportion of plastid genes utilize GC-rich codons, which are preferentially recognized by the cytoplasmic tRNA pools of germinating cells. This GC-bias-driven codon optimization allows leucoplast mRNAs to be efficiently translated even outside their original organelle context, particularly in cells with high metabolic activity, such as those found in root tips and differentiating meristems.

[0009] Furthermore, AEM2 mRNAs possess functional structural elements that support smooth translation in the cytosolic environment. These include a native 5' cap structure, conserved ribosome-binding-like motifs in the 5' UTRs, and a polyadenylated 3' region, which together ensure stable ribosome binding and prolonged transcript persistence.

[0010] This molecular adaptation to the translational apparatus of the host cell forms the basis for the high biological activity of AEM2 mRNA. It allows the efficient synthesis of plastid-encoded proteins in non-photosynthetic tissues, thereby modulating hormonal and morphogenetic signaling pathways without requiring genetic integration of the sequences into the genome of the target plant.

[0011] 1. Composition and source of AEM2 mRNA:

[0012] • Starting material: AEM2 mRNA is isolated from leucoplasts in germinating seeds, where it plays a fundamental role in promoting auxin synthesis and the production of auxin-related proteins.

[0013] • Primary function: AEM2 mRNA enhances the localization and activity of auxin transport proteins (e.g., PIN and AUX1 / LAX) and optimizes auxin transport based on intrinsic cellular polarity rather than gravity-dependent cues. This mechanism compensates for impaired statolith function in microgravity. 2. Mechanism of action of AEM2 mRNA in microgravity:

[0014] Improved PIN localization and intrinsic polarity

[0015] - Fig. 1

[0016] • PIN protein optimization: AEM2-m RNA promotes the stability and localization of PIN proteins along the cell's intrinsic polarity axis, anchoring these proteins in positions that maintain directed auxin flow.

[0017] • Formation of internal auxin gradients: By establishing intrinsic polarity, AEM2 mRNA enables the formation of auxin gradients that support the orientation of roots and shoots, even in the absence of gravitational cues.

[0018] Cellular uptake and distribution of AEM2 mRNA

[0019] Following application via the leaf surface in a plant biopolymer matrix or via root uptake from the granules, AEM2 mRNA preferentially enters target cells via plasmodesmata and vesicular transport processes. It is assumed that endocytosis-like mechanisms and extracellular vesicles (EVs) are involved, releasing the mRNA into the apoplast space and transferring it into the cytoplasm of the recipient cells. Alternatively, interaction with microbial symbionts (e.g., mycorrhizae or rhizobacteria) can result in a horizontal gene transfer-like exchange of RNA, enabling transfer to root cells. These transport pathways ensure efficient distribution of AEM2 mRNA within the tissue and guarantee its entry into metabolically active cells.

[0020] Securing translational activity

[0021] The AEM2 mRNA exhibits typical eukaryotic 5' cap structures (m 7The mRNA sequences feature a GpppN nucleotide and a poly(A) tail at the 3' end, optimizing stability and ribosome binding. These structural features enable translation by endogenous plant 80S ribosomes. Since the mRNA sequences originate from leucoplasts, they possess an optimized codon utilization profile compatible with cytoplasmic tRNA pools, ensuring efficient protein synthesis in target cells. The translation products subsequently interact with intracellular membranes and signaling complexes, particularly in the endoplasmic reticulum and plasma membrane, thus influencing intracellular signaling pathways.

[0022] At the molecular level, the expression of AEM2-encoded proteins leads to the activation of serine / threonine kinases of the PID / WAG family, which control the polarization and relocalization of PIN transporters. Simultaneously, inhibition of protein phosphatase 2A (PP2A) is observed, which stabilizes the phosphorylated, active form of the PIN proteins and maintains auxin polarity. This modulation of the kinase-phosphatase balance is considered a central element of gravity-independent auxin regulation.

[0023] Secondary signals and stabilization of auxin polarity

[0024] Additionally, treatment with AEM2 mRNA induces a moderate formation of reactive oxygen species (ROS), which act as secondary signals for the activation of Ca 2+β-dependent kinases (CDPKs) contribute to this process. The resulting phospholipid gradients in the plasma membrane enhance the formation of stable auxin flow directions by supporting the anchoring of PIN proteins in polarized domains. These mechanisms act synergistically to establish intrinsic cell polarity and compensate for the absence of gravitropic stimuli under microgravity conditions.

[0025] The combination of these processes – uptake, translation, signal activation, and auxin flux stabilization – constitutes the central operating principle of AEM2 technology. This ensures that directed auxin distribution is maintained even in microgravity, resulting in high root direction stability and stable plant morphogenesis.

[0026] Simulation of a gravity-independent auxin flow

[0027] - Fig. 2

[0028] • Pseudo-gravity cues: Enhanced intrinsic polarity in AEM2 mRNA-treated cells simulates gravity-dependent auxin redistribution, directing auxin flow to the lower side of root cells. This gradient effect allows roots to grow in an organized direction, mimicking gravitropic responses on Earth. • Auxin flow consistency: Optimized interaction between PIN and AUXI / LAX transporters supports the stability and directional dependence of auxin flow, essential for structured root and shoot growth.

[0029] Reduction of random growth patterns

[0030] - Fig. 3

[0031] • Consistency of root alignment: Treatment with AEM2 mRNA reduces irregular growth patterns by directing auxin onto predictable pathways, thus creating a stable alignment for root development.

[0032] • Improved directional orientation of root tips: Root tips exhibit more uniform downward growth, reducing the randomness often seen in plants grown in zero gravity.

[0033] Supporting exploratory root growth

[0034] - Fig. 4

[0035] • Auxin-directed elongation: AEM2 mRNA enhances the auxin gradients required for exploratory root growth, enabling roots to grow in a specific direction, improving resource-searching behavior, and promoting a more functional root system.

[0036] Extension of the detailed description of the invention

[0037] The AEM2 mRNA used comprises a spectrum of plastid transcripts from leucoplasts of germinating seeds, typically ranging in length from 0.5 kb to 3.0 kb. These transcripts represent functional mRNA populations, particularly sequences encoding enzymes of auxin synthesis (e.g., anthranilate synthase alpha subunit 1 [ASA1] and tryptophan synthase beta subunit 1 [TSB1]), proteins of lipid biosynthesis (such as acyl carrier protein acpP), and regulatory proteins with kinase activity (e.g., PINOID-like kinases, PID-like). These mRNAs are characteristic of the plastid regulation of cellular polarity and auxin homeostasis. They contribute to the establishment of stable auxin gradients by influencing the localization and activity of auxin transport proteins, especially the PIN and AUX1 / LAX families, via plastid-nucleus communication pathways.

[0038] For the experimental characterization of AEM2 mRNA, an RNA sequencing method (RNA-Seq) can be used to determine the specific transcript profiles of plastid genes from leucoplasts of germinating seeds. In addition, quantitative real-time PCR (qRT-PCR) can be used to verify the expression levels of selected transcripts, and proteomic analysis (LC-MS / MS or Western blot) can be employed to identify the translationally active proteins.

[0039] AEM2 mRNA is preferably isolated from seedling leucoplasts. Leucoplasts are enriched by density gradient centrifugation (e.g., Percoll gradients with graded density). RNA extraction is performed according to established procedures, preferably using a TRIzol reagent or LiCI precipitation protocol to ensure high integrity and purity of the isolated mRNA.

[0040] The described parameters enable reproducible production of the AEM2 mRNA component and ensure the experimental reproducibility of the functionality claimed in this invention, particularly with regard to gravity-independent auxin modulation.

[0041] Extended applications for reducing growth disorders In addition to stabilizing auxin transport and auxin orientation, AEM2-mRNA addresses several other growth disorders resulting from impaired statolith function in weightlessness:

[0042] 1. Improved function of the root cap and formation of root hairs:

[0043] • Improved integrity of the root cap: AEM2 mRNA stabilizes auxin flow in the root cap, thereby maintaining root tip protection and directed growth. • Uniform development of root hairs: Stable auxin gradients promote root hair formation and improve the efficiency of water and nutrient uptake under microgravity.

[0044] Restoration of root meristem activity and cell division:

[0045] • Active meristem maintenance: Auxin gradients supported by AEM2 mRNA keep the root meristem active and promote uniform cell division and elongation, which are essential for root growth.

[0046] • Improved root growth rate: This feature enables plants to achieve normal root growth and biomass accumulation under microgravity conditions.

[0047] Improved formation of lateral roots:

[0048] • Controlled branching: The redistribution of auxin supports the formation of lateral roots, resulting in a more branched root network that improves nutrient uptake.

[0049] • Extended root architecture: Increased formation of lateral roots gives the plant access to a broader resource base, improving resilience and growth stability.

[0050] Optimized leaf positioning and stem development:

[0051] • Leaf orientation for optimal light uptake: By maintaining stable auxin gradients, AEM2 mRNA supports uniform leaf positioning, thus maximizing the efficiency of photosynthesis.

[0052] • Increased stem growth: The improved auxin transport leads to regular stem elongation and structural integrity, which is crucial for maintaining plant shape and function.

[0053] Balanced hormonal responses and reduced oxidative stress: • Stress resilience: The role of AEM2 mRNA in stabilizing auxin flow mitigates oxidative stress in weightlessness, improves cellular resilience and reduces the level of reactive oxygen species (ROS).

[0054] • Hormonal balance: The restored auxin flow supports the interaction with other growth hormones and reduces the hormonal imbalance common in weightlessness.

[0055] 6. Improved flowering and reproductive success:

[0056] • Support of flower formation: AEM2 mRNA enables stable auxin gradients, which are necessary for flower and bud formation, thus ensuring uniform reproductive development.

[0057] • Improved seed and fruit production: Plants treated with AEM2 mRNA develop fruits and seeds with greater predictability, which supports the germination rate of the seeds for future plant generations.

[0058] Industrial applicability

[0059] This invention opens the door to sustainable agriculture in space habitats by enabling uniform, organized plant growth and correcting critical growth deficiencies caused by impaired statolith function. Through the ability of AEM2 mRNA to enhance intrinsic cellular polarity and auxin transport, this technology allows plants to develop functional root systems, balanced hormonal responses, and reliable reproductive cycles, which are essential for long-term plant productivity in microgravity.

[0060] AEM2 technology can be integrated into scalable, controlled systems that ensure reproducible application. In particular, it can be implemented in standardized pouring or spraying systems operating under strictly controlled environmental conditions, such as those required in closed habitats, for example, on the International Space Station (ISS) or future facilities like the Lunar Gateway. This integration into automated or semi-automated application processes makes the technology accessible for both precision agriculture and extraterrestrial production systems.

[0061] The applications described range from small experimental setups to commercial production systems and fully enclosed habitats, with the technology enabling reproducible, efficient and safe implementation of biological activators at all times.

[0062] Fig. 1

[0063] Improved PIN localization and intrinsic polarity

[0064] Auxin gradient consistency

[0065] Chart legend and details

[0066] 1. PIN protein localization and stability

[0067] • Earth's gravity (control): 90% - Represented by slashes ( / / )

[0068] • Untreated microgravity: 35% - Represented by slashes (\)

[0069] • AEM2 mRNA-treated microgravity: 70% - Represented by dotted patterns (o)

[0070] 2. Auxin gradient consistency

[0071] • Earth gravity (control): 85% - Represented by a slash pattern ( / / ) • Untreated microgravity: 40% - Represented by a slash pattern (\) • AEM2 mRNA-treated microgravity: 75% - Represented by a dotted pattern (o)

[0072] Result description: PIN protein localization and stability

[0073] overview

[0074] • Earth's gravity (control): Achieved the highest level of PIN protein stability and localization with a value of 90%.

[0075] • Untreated microgravity: Showed a significant drop to 35%, indicating that PIN protein localization is highly dependent on gravitational signals. • AEM2 mRNA-treated microgravity: Improved to 70%, reflecting a substantial improvement in PIN protein stability and localization with AEM2 mRNA, although not quite matching Earth's gravity conditions.

[0076] Interpretation and explanation

[0077] • Why PIN localization is important: PIN proteins are essential for directed auxin transport, establishing cellular polarity and supporting the formation of auxin gradients, which are crucial for root and shoot orientation. Under Earth's gravity, statoliths control PIN localization, helping to position these proteins on the plasma membrane to direct auxin flow in response to gravitational signals.

[0078] • Effect of microgravity: In microgravity, the lack of gravitational cues prevents proper PIN localization, reducing the effectiveness of auxin transport and leading to irregular growth patterns. The low stability value (35%) under untreated microgravity conditions reflects this deficiency, as PIN proteins cannot maintain their usual positions, resulting in disrupted auxin transport.

[0079] • Effects of AEM2 mRNA: AEM2 mRNA enhances intrinsic cellular polarity and helps position PIN proteins along the cell's natural polarity axis. This intrinsic regulation partially compensates for the lack of gravity, resulting in PIN localization stability of 70%. While this value does not fully match the force of gravity, it demonstrates that AEM2 mRNA significantly restores PIN protein function in microgravity and enables more directed auxin flow even without the influence of gravity.

[0080] Result description: Auxin gradient consistency

[0081] Overview • Earth's gravity (control): Maintaining high auxin gradient stability with a value of 85%, which shows a robust auxin distribution along typical growth axes.

[0082] • Untreated microgravity: A lower auxin gradient stability of 40% was indicated, suggesting an inconsistent auxin distribution without gravitational evidence.

[0083] • AEM2 mRNA-treated microgravity: Increased auxin gradient stability to 75%, suggesting that AEM2 mRNA helps maintain directed auxin flow by creating internal gradients that mimic gravity conditions on Earth.

[0084] Interpretation and explanation

[0085] • Why the consistency of the auxin gradient is important: Auxin gradients are essential for directed cell expansion and cell growth. In roots, auxin gradients influence cell elongation by directing root tips downwards and supporting the integrity of the root cap. These gradients also influence shoot growth by orienting the plant upwards and facilitating leaf positioning for optimal light uptake.

[0086] • Effects of microgravity: Without the influence of gravity, auxin gradients become inconsistent, leading to random growth patterns. The untreated microgravity state, with an auxin gradient stability of 40%, reflects this challenge, as the cells lack the internal alignment required for organized growth.

[0087] • Effects of AEM2 mRNA: By enhancing PIN localization and cellular polarity, AEM2 mRNA generates a pseudo-gravitational effect, enabling cells to establish and maintain auxin gradients even in microgravity. The resulting auxin gradient stability of 75% in AEM2 mRNA-treated plants demonstrates that these gradients can be maintained with near-Earth-like consistency, thus providing a stable framework for directed root and shoot growth.

[0088] Overall result explanation: The results suggest that AEM2 mRNA partially restores directed growth mechanisms in plants by enhancing intrinsic polarity and PIN localization, even without gravitational cues. This result is significant for the following reasons:

[0089] 1. Establishment of pseudo-gravitational stimuli: AEM2 mRNA enables cells to rely on intrinsic polarity for PIN protein positioning, creating internal auxin gradients that guide directed growth similar to gravity-based responses. This internal organization helps compensate for the lack of external gravitational stimuli. 2. Support of functional root and shoot orientation: With more stable PIN localization and auxin gradients, AEM2 mRNA-treated plants can establish organized root and shoot orientation. This minimizes the irregular growth typical of microgravity, allowing roots to grow downwards and shoots upwards, which is crucial for nutrient uptake and light exposure.

[0090] 1. Potential for space agriculture: These results highlight the potential of AEM2 mRNA to stabilize plant growth in space environments, where uniform root and shoot alignment is essential for sustainable agriculture. By restoring auxin transport mechanisms in microgravity, AEM2 mRNA treatment could enable plants to thrive in space-based habitats, thus contributing to long-term crop production.

[0091] Summary

[0092] The results show that AEM2 mRNA can significantly improve the efficiency of auxin transport and cellular polarity under microgravity conditions.

[0093] Although gravity in space is not identical to that on Earth, plants treated with AEM2 mRNA achieve PIN localization and auxin gradient consistency values ​​close enough to ensure functional growth orientation. This ability to restore directed growth without gravity signals underscores the value of AEM2 mRNA in space agriculture and offers a viable solution to the challenges of cultivating plants in microgravity.

[0094] Fig. 2

[0095] Simulation of gravity-independent auxin flow

[0096] Consistency of auxin flow

[0097] Legend and details about the graphic

[0098] 1. Pseudo-gravity evidence

[0099] • Earth's gravity (control): 99% - Represented by slashes ( / / )

[0100] • Untreated microgravity: 30% - Represented by inverted slashes (\)

[0101] • AEM2 mRNA-treated microgravity: 72% - Represented by dotted patterns (o)

[0102] This graph illustrates the efficacy of AEM2 mRNA treatment in compensating for the absence of gravitational cues in microgravity. While untreated microgravity conditions result in a low degree of pseudo-gravitational cues (30%), AEM2 mRNA treatment restores 72% of these cues, approaching Earth's gravity conditions by 99%. This significant improvement supports the hypothesis that AEM2 mRNA establishes internal directional cues in root cells that simulate gravitational effects.

[0103] 2. Auxin flow consistency

[0104] • Gravity (control): 97% - Represented by slashes ( / / )

[0105] • Untreated microgravity: 38% - Represented by slashes (\) • AEM2 mRNA-treated microgravity: 77% - Represented by dotted patterns (o)

[0106] This graph shows that AEM2 mRNA treatment increases the stability of the auxin gradient to 77% of Earth's gravity, compared to only 38% under untreated microgravity conditions. The higher consistency of auxin flux in AEM2 mRNA-treated plants suggests a well-structured auxin distribution even without gravity, supporting more predictable and organized growth patterns.

[0107] Explanation of the results

[0108] The significant improvement in both pseudo-gravity signals and auxin flow consistency following treatment with AEM2 mRNA can be attributed to the following mechanisms:

[0109] 1. Establishing intrinsic polarity for directed signals

[0110] Mechanism: AEM2 mRNA enhances the intrinsic cellular polarity that controls the positioning of PIN proteins in root cells. Under Earth's gravity, statoliths (small organelles that respond to gravity) would normally control this positioning, but in their absence, AEM2 mRNA supports an internal polarity axis. This axis allows auxin to flow in a way that mimics gravitational stimuli.

[0111] Result: By generating these intrinsic directional cues, AEM2 mRNA enables the accumulation of auxin in specific regions of the root, particularly on the underside, even under microgravity. This pseudo-gravity effect, reflected in the 72% pseudo-gravity cue level in AEM2 mRNA-treated plants, promotes better-organized root orientation and improved root growth.

[0112] 2. Improved PIN localization and stability mechanism

[0113] Mechanism: PIN proteins play a central role in auxin transport, and their positioning on the cell membrane determines the direction of auxin flow. AEM2 mRNA enhances the stability and localization of PIN proteins by anchoring them along the cell's intrinsic polarity. This arrangement promotes directed auxin flow independent of gravity. Result: The improved localization and stability of PIN proteins leads to a more uniform auxin flow, as demonstrated by the 77% auxin flow consistency observed in AEM2 mRNA-treated plants. This stability is crucial for maintaining predictable auxin gradients, which control cell elongation and root curvature, preventing the irregular growth often seen under microgravity conditions.

[0114] erung of the mechanism of the AUX1 / LAX transporter function

[0115] Mechanism: AUX1 / LAX transporters help maintain auxin homeostasis by facilitating auxin influx into cells. Treatment with AEM2 mRNA appears to optimize the function of these transporters in conjunction with PIN proteins, balancing auxin levels across cell junctions. This interaction is essential for a consistent, gravity-independent auxin flow.

[0116] Result: The combined function of the PIN and AUX1 / LAX transporters, supported by AEM2 mRNA, enables cells to maintain auxin gradients similar to those driven by gravity. This optimization improves the overall consistency of auxin distribution and stabilizes root-shoot alignment, reflected in higher values ​​for auxin flux consistency.

[0117] I am responsible for the gravity-dependent mechanism of auxin redistribution

[0118] Mechanism: Under normal gravity conditions, auxin is redistributed to the lower side of the plant's root cells, causing the root to grow downwards. Treatment with AEM2 mRNA compensates for the absence of this gravitational redistribution by establishing a directed auxin flow based solely on cellular polarity.

[0119] Result: This pseudo-gravitational effect directs auxin to specific areas within the root, thus simulating the soil-based gravitropic response that promotes downward root growth. The graph shows a clear advantage of AEM2 mRNA-treated plants over untreated microgravity controls and supports the theory that AEM2 mRNA can act as a substitute for gravity in auxin redistribution.

[0120] conclusion

[0121] The results indicate that treatment with AEM2 mRNA significantly improves both the simulation of gravity-like stimuli and the consistency of auxin flow in microgravity environments. By enhancing intrinsic polarity, stabilizing PIN localization, and optimizing transporter function, AEM2 mRNA enables plants to maintain structured growth patterns even without the influence of gravity. These effects are crucial for promoting organized root and shoot orientation in space-based agriculture and offer a promising solution to the challenges of crop cultivation in microgravity.

[0122] This improvement has direct implications for space agriculture, as it suggests that AEM2 mRNA plays a key role in supporting sustainable plant growth and food production in space.

[0123] Space habitats can be played.

[0124] Fig. 3

[0125] Reduction of random growth patterns

[0126] Directional dependence of the root tip during the course of current

[0127] Legend - Earth's gravity (control)

[0128] Untreated microgravity

[0129] — ( - AEM2-mRNA-treated microgravity

[0130] 1. Consistency of root orientation over time

[0131] X - Weeks Y - Root Orientation Consistency

[0132] 2. Improved alignment of root tips over time

[0133] X weeks

[0134] Y - Root tip direction determination

[0135] 1. Consistency of root orientation over time

[0136] • Gravity (control): Starts at 95% and rises to 98%, representing the stable and consistent root orientation supported by gravity. This high level of consistency is due to the influence of gravity on auxin distribution, which directs the roots downwards.

[0137] • Untreated microgravity: Shows a gradual decrease from 55% to 47%, illustrating an increase in irregular growth patterns due to the lack of gravitational cues.

[0138] • AEM2 mRNA-treated microgravity: Starts at the same level as untreated microgravity (55%), but improves continuously, reaching 676% in week 1. This improvement suggests that AEM2 mRNA stabilizes root orientation by improving internal auxin transport pathways and reducing the randomness of root growth even without gravity.

[0139] 2. Improved alignment of root tips over time

[0140] • Gravity (control): Remains consistently high between 96% and 98%, indicating that the root tips reliably grow downwards under the influence of gravity.

[0141] • Untreated microgravity: Decreases from 50% to 41%, reflecting the random orientation of root tips due to the lack of directional cues from gravity.

[0142] • AEM2 mRNA-treated microgravity: Starting at 50%, similar to the untreated microgravity group, but steadily increasing to 75% by week 6. This improvement in directional orientation demonstrates that AEM2 mRNA treatment promotes more uniform downward root tip growth by providing internal pseudogravity cues through optimized auxin flow.

[0143] Detailed description of the graphic

[0144] These figures illustrate the impact of AEM2 mRNA treatment on reducing random growth patterns in two areas:

[0145] Root orientation consistency and root tip direction determination over a six-week period. Each graph compares three groups under different growth conditions:

[0146] 1. Earth's gravity (control): Shows stable and predictable growth patterns with high consistency in root orientation and direction determination based on natural gravitational cues.

[0147] 2. Untreated microgravity: Shows a decline over time in both the consistency of root alignment and the directional orientation of the root tips, reflecting the disorganized and irregular growth typical of microgravity when gravity-dependent auxin signals are absent.

[0148] 3. AEM2 mRNA-treated microgravity: Starting with values ​​similar to those of untreated microgravity, but showing a steady improvement in the consistency of root orientation and the directional dependence of root tips. This suggests that AEM2 mRNA treatment can partially compensate for the lack of gravity, thereby enabling a more organized root growth pattern.

[0149] Explanation of the results

[0150] These results show that AEM2 mRNA treatment significantly improves the consistency of root orientation and the directional dependence of the root tip in microgravity. This improvement is attributed to the stabilizing effect of AEM2 mRNA on auxin transport: 1. Control of auxin flow along predictable pathways: AEM2 mRNA improves the stability and localization of auxin transport proteins (PIN and AUX1 / LAX), which direct auxin along the cell's intrinsic polarity. This intrinsic control partially compensates for gravitational signals and generates internal auxin gradients that promote directed root growth.

[0151] 2. Generation of pseudo-gravity signals: In the absence of gravity, treatment with AEM2 mRNA helps to simulate gravity-dependent auxin redistribution. By enhancing intrinsic polarity and creating stable auxin gradients, the treatment enables root cells to orient themselves downwards and grow in a more controlled manner, similar to terrestrial growth patterns.

[0152] 3. Improved root tip orientation: The root tips of plants treated with AEM2 mRNA are more uniformly downward-oriented than those of untreated plants under microgravity conditions. This improvement supports structured growth and resource uptake, as an organized root architecture is essential for nutrient and water uptake.

[0153] Importance for plant growth in space

[0154] These results are of great importance for space agriculture, as they demonstrate that AEM2 mRNA treatment can partially restore the organized root growth and orientation required for effective resource absorption and plant stability in microgravity. By supporting uniform root orientation and minimizing irregular growth, AEM2 mRNA offers a promising solution for cultivating plants in space habitats, where predictable and stable growth patterns are essential for sustainable crop production.

[0155] Fig. 4

[0156] AEM2 mRNA support for exploratory root growth

[0157] Exploratory root growth over time

[0158] Explanation of the results on the growth of the exploration roots Legend - — <— Earth's gravity (control)

[0159] — Untreated microgravity

[0160] ■ “AEM2-mRNA-treated microgravity

[0161] X weeks

[0162] Y - Exploratory root growth measurement (%)

[0163] The graph shows how different growth conditions—Earth gravity (control), untreated microgravity, and AEM2 mRNA-treated microgravity—affect the growth of the plant's exploratory roots over a six-week period. The results demonstrate a clear advantage of AEM2 mRNA treatment in supporting extensive root exploration, particularly under the stress of microgravity.

[0164] 1. Earth's gravity (control)

[0165] • Result: Exploratory root growth under Earth gravity conditions starts at 23% and follows a steady upward trend, reaching 639% in week 6.

[0166] • Explanation: Under Earth's gravity, plants receive natural gravitational signals that direct their roots downwards and support a stable root architecture. Because gravity already assists root orientation and nutrient uptake, plants under Earth's gravity do not exhibit extensive exploratory growth. Instead, they maintain a moderate root exploration pattern without the need for strong root spread.

[0167] • Significance: This moderate growth serves as a control mechanism, indicating how plants naturally limit root exploration when there are ample directional cues and stable resources.

[0168] 2. Untreated microgravity • Result: Untreated plants in microgravity begin with similar exploratory growth to plants treated with AEM2 mRNA (17%), but experience a steady decline, ending at 10% in week 6.

[0169] • Explanation: In weightlessness, the lack of gravitational stimuli causes root growth to become disoriented and irregular. Without orientation, roots are unable to effectively explore their environment or find a reliable orientation. As a result, untreated plants exhibit limited root spread and reduced exploratory behavior, negatively impacting their ability to access nutrients and water.

[0170] • Significance: This finding underscores the challenge microgravity poses to plant growth, as plants lose their natural root orientation mechanisms. The absence of exploratory growth suggests that the plant is unable to adapt effectively to weightlessness, resulting in inefficient resource acquisition.

[0171] Result of AEM2 mRNA treatment under weightlessness

[0172] • Starting from the same baseline as untreated zero-gravity plants (17%), AEM2 mRNA-treated plants show a significant increase in exploratory growth, reaching 668% in week 1 - a dramatic improvement over both Earth gravity and untreated zero-gravity conditions.

[0173] • Explanation: AEM2 mRNA treatment enhances the plant's internal auxin transport pathways and promotes intrinsic cellular polarity, which compensates for the lack of gravity. This treatment strengthens root tip proliferation, a crucial factor for increased root branching and expansion. Furthermore, the extensive root tip development induced by AEM2 mRNA treatment enables unusually high levels of mycorrhizal symbiosis. This symbiotic relationship increases the efficiency of nutrient and water uptake and prompts the plant to extend its roots further and explore the growing medium more effectively. The steady increase in exploratory growth reflects the plant's adaptive response to resource scarcity, facilitated by the enhancement of root development mechanisms through AEM2 mRNA.

[0174] • Significance: This result is of great importance for space-based plant breeding, as it shows that treatment with AEM2 mRNA can overcome the disorientation caused by microgravity. The treatment enables plants to maintain robust root exploration and resource-seeking behavior even under the extreme challenge of weightlessness.

[0175] Relevance of the results for space agriculture

[0176] The clear benefits of AEM2 mRNA treatment under microgravity have significant implications for sustainable agriculture in space environments:

[0177] 1. Improved resource uptake in confined environments: In space habitats where soil volume and nutrient availability are limited, plants with high exploratory root growth have a better chance of accessing the limited resources available. The enhanced root tip development and mycorrhizal symbiosis supported by AEM2 mRNA treatment are crucial for maximizing resource absorption in a confined environment.

[0178] 2. Compensation for gravity-induced disorientation: By promoting intrinsic cellular polarity and stable auxin gradients, AEM2 mRNA treatment effectively simulates the orienting influence of gravity. This allows plants to explore their environment even in the absence of gravity and maintain a growth pattern similar to Earth's conditions in terms of root extension and nutrient uptake.

[0179] 3. Sustainable Plant Growth for Long-Term Missions: Robust root systems that enable high exploratory growth are essential for the long-term cultivation of crops in space. The AEM2 mRNA treatment allows plants to develop resilient root architectures that can adapt to non-terrestrial environments and support ongoing food production in future space habitats. Conclusion

[0180] The results in this table demonstrate that treatment with AEM2 mRNA offers transformative benefits for plants grown in microgravity. By promoting exploratory root growth and mycorrhizal symbiosis, AEM2 mRNA treatment helps plants overcome disorientation caused by microgravity and supports a more adaptable, resource-efficient root system. This capability is essential for successful plant breeding in space environments, making AEM2 mRNA a powerful tool for future space agriculture and long-duration space missions.

[0181] Summary of the invention

[0182] The present invention uses AEM2 mRNA, derived from leucoplasts in germinating seeds, to enable intrinsic cellular polarity and enhance auxin transport pathways independent of gravity. Leucoplasts represent a non-photosynthetic, yet metabolically highly active form of plastids that play a central role in the hormonal and morphogenetic control of growth, particularly during the early germination phase. These transcripts are found in the

[0183] These mRNAs are particularly stable and translationally active during the postgerm inative developmental phase because they are not regulated by light-dependent signaling pathways or photo-oxidative stress. This results in a unique mRNA profile that is geared towards the hormonal activation and differentiation of early seedling structures. Isolation of these mRNAs from leucoplasts thus provides a highly purified, non-photosynthetic information source directly involved in establishing cell polarity and auxin transport. These mRNAs preferentially encode proteins that influence auxin biosynthesis, polar transport, and cytoskeletal organization, and are therefore particularly well-suited to support gravity-independent growth processes.By optimizing the localization and activity of PIN and AUX1 / LAX proteins, AEM2 mRNA enables stable auxin gradients that support directed root growth, organized shoot orientation, and overall plant architecture under microgravity conditions.

[0184] Furthermore, it has been found that these same mechanisms are also effective under terrestrial conditions, particularly when plants exhibit inactive or dormant growth states due to abiotic stress factors (heat, drought, cold, nutrient deficiency). The application of AEM2 mRNA enables the reactivation of auxin flow and a systemic restoration of meristem activity in these cases.

[0185] AEM2 mRNA also mitigates various growth deficiencies, including impaired root cap function, reduced lateral root formation, irregular leaf positioning, and inhibited meristem activity, which are frequently observed when statolith function is impaired.

[0186] In simulated microgravity systems, the application of AEM2 mRNA to model plants showed a significantly increased auxin polarization and root orientation, confirming the technical reproducibility of the invention.

[0187] This approach is crucial for promoting sustainable agriculture in space, as it allows plants to develop stable root systems, efficient nutrient uptake mechanisms, and balanced growth cycles, even in the absence of gravity guidance.

Claims

Patent claims 1. Use of exogenous mRNA isolated from leucoplasts of germinating plants (AEM2 mRNA) to increase directed auxin transport under reduced gravity conditions, characterized in that the mRNA includes sequences encoding proteins of the PIN and AUX1 / LAX transport systems or their regulatory kinases, thereby stabilizing intrinsic cellular polarity and maintaining directed auxin translocation.

2. Use of AEM2 mRNA to initiate stable auxin gradients in plant cells under conditions of reduced or absent Gravitation, characterized in that the mRNA induces the formation of a functional polarity axis, which enables auxin-mediated orientation of root and shoot growth in the absence of a gravity vector.

3. Use of AEM2 mRNA in plant tissues to reduce stochastic or disordered growth directions in the absence of gravitational signals, characterized in that the mRNA-mediated expression of auxin transport and polarization factors stabilizes root orientation and maintains a coherent growth vector.

4. Use of AEM2 mRNA isolated from leucoplasts of germinating plants to promote root extension and branching in plants under reduced or absent gravity, characterized in that the expressed transcripts generate stable auxin gradients which enable directed growth towards local nutrient concentrations and microbial soil microgradients.

5. Use of AEM2 mRNA for the prevention or reduction of statolith-related growth deficiencies in plants under microgravity, characterized in that the translated proteins cause increased activity of plastid signaling pathways and cellular polarity markers, leading to improved root cap function, increased root hair formation, increased This leads to meristem activity, uniform lateral root development, optimized leaf position and regulated shoot elongation.

6. Use of AEM2 mRNA to stabilize cellular redox status and maintain hormonal homeostasis in plants under reduced gravity conditions, characterized in that the translation of the mRNA activates enzymes and regulatory proteins of the auxin, cytokinin and abscisic acid signaling pathways, thereby reducing oxidative stress and maintaining the structural integrity of cell membranes, chloroplasts and cytoskeleton.

7. Use of exogenous mRNA (AEM2) isolated from leucoplasts of any seedling species to support gravity-independent directed auxin transport, characterized in that the applied mRNA compensates for growth and development deficits caused by the absence of gravity-dependent signaling.

8. Use of the AEM2 mRNA according to one of the preceding claims for restoring auxin flow, meristem activity and flowering capacity in terrestrial plants under abiotic stress or dormancy, characterized in that the application of the AEM2 mRNA triggers a plastid-nuclear feedback (retrograde signaling) which leads to a systemic reactivation of growth within a short time after application and restores intracellular polarity and directed auxin transport.

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