Method for increasing plant growth using phyto-initiator mrnas in plant cells

Abstract

The invention relates to a method for increasing plant growth by delivering phyto-initiator mRNAs derived from chloroplasts of seedlings. The mRNAs may either be translated directly in chloroplasts or first translated in the cytosol and subsequently imported into the chloroplasts in the form of proteins. In both cases, the resulting proteins activate hormonal signalling pathways, increased protein synthesis, and bring about a significant enhancement of root biomass, shoot number and hypocotyl growth. The invention also relates to formulations and application systems such as granules, sprays or injections which enable efficient uptake and activity of the mRNAs.

Description

[0001] October 15, 2025

[0002] Methods for increasing plant growth using phytoinitiator mRNAs in plant cells

[0003] Description

[0004] The invention relates to a method for increasing plant growth based on so-called phytoinitiator mRNAs obtained from newly formed chloroplasts in seedlings.

[0005] Background of the invention:

[0006] Conventional methods for promoting plant growth, such as chemical fertilizers, are often insufficient to fully optimize plant development, particularly at the molecular level. Recent advances in molecular biology demonstrate the potential of manipulating chloroplast mRNA to enhance plant growth and resilience.

[0007] This invention introduces the concept of phytoinitiator mRNAs derived from newborn chloroplasts of a germinating seed to enhance growth by optimizing protein synthesis through ribosome competition and natural regulatory mechanisms. These mRNAs contain transcripts for enzymes, regulatory proteins, and signaling molecules characteristic of the physiological state of the germination phase. The application of these phytoinitiator mRNAs to other plant cells reprograms plastid and nuclear signaling pathways, thereby increasing the biosynthesis of growth-promoting proteins.

[0008] In contrast to previous strategies, this approach focuses on direct mRNA-based regulation within chloroplasts and offers a more sustainable and biological alternative for improved plant growth. Summary of the invention:

[0009] The invention relates to a method for influencing plant growth by external supply of phytoinitiator mRNAs obtained from chloroplasts of freshly germinated seeds.

[0010] In a first embodiment, the mRNA is introduced into living plant tissue by microinjection and immediately translated into chloroplasts. The mRNAs exhibit dissociation constants (Kd) of 10⁻⁵. 8 - 10' 9 mol L -1 compared to plastid 70S ribosomes, thereby leading to efficient synthesis of growth-promoting proteins that reprogram plastid redox and signaling networks.

[0011] In a second embodiment, the mRNA is administered via leaf or soil application, translated in the cytosol and produces proteins there that activate or enhance hormonal and metabolic signaling pathways.

[0012] Both mechanisms result in a plastid-nuclear reorganization that sustainably optimizes the transcription of growth-relevant genes and plant-wide hormone homeostasis.

[0013] Plants treated with phytoinitiator mRNAs show:

[0014] • 15-30% increase in root mass

[0015] • 10-30% increase in the number of drives

[0016] • 5-20% increase in hypocotyl growth

[0017] This method is particularly useful in controlled environments such as greenhouses or vertical farms, where natural conditions can hinder optimal growth processes.

[0018] 1. Detailed description of the invention:

[0019] A. Phytoinitiator mRNA in Chloroplast Ribosome Optimization. Phytoinitiator mRNAs, a group of specialized mRNAs derived from newborn chloroplasts of germinating seeds, enhance protein synthesis by optimizing ribosomal competition within the chloroplast. When these mRNA sequences are introduced into chloroplast ribosomes, they increase ribosomal efficiency, thus enabling a higher translation rate for important growth-related proteins. Unlike the cytoplasmic nonsense-mediated decay (NMD) pathway, there is no direct equivalent in chloroplasts. Therefore, the mechanism in chloroplasts focuses on ribosome allocation and competition. Phytoinitiator mRNAs bind to the ribosomes of chloroplasts with a higher affinity than native mRNAs, enabling optimized protein production associated with root, shoot, and hypocotyl development.

[0020] In the first embodiment, the mRNA is introduced into living plant tissue by microinjection and immediately translated into chloroplasts. The mRNAs exhibit dissociation constants (Kd) of 10 -8 — 10 -9 mol L -1 compared to plastid 70S ribosomes, thereby leading to efficient synthesis of growth-promoting proteins that reprogram plastid redox and signaling networks.

[0021] In the second embodiment, the translation of exogenous mRNAs takes place in the cytosol of the plant cell. The proteins produced in this process can contain import sequences that enable their transport into the chloroplasts and activate or enhance hormonal and metabolic signaling pathways. Import occurs via the known translocation systems of the chloroplasts (TOC / TIC complexes), after which the proteins exert their growth-promoting functions within the chloroplasts. Thus, the invention encompasses both direct translation in chloroplast ribosomes and translation in the cytosol with subsequent protein import.

[0022] Both mechanisms result in a plastid-nuclear reorganization that sustainably optimizes the transcription of growth-relevant genes and plant-wide hormone homeostasis.

[0023] Mechanism of action:

[0024] Following application, the exogenous phytoinitiator mRNAs enter the cytosol of the target cells, where they are translated by cellular ribosomes. The proteins produced in this process act as enzymes, regulators, or signals and contain transit peptides that enable their targeted import into chloroplasts. Within the chloroplasts, these proteins induce local metabolic reprogramming, particularly through alterations in the redox potential, ATP / NADPH ratios, and reactive oxygen species (ROS). These shifts activate plastid-nuclear signaling pathways (retrograde signaling) via messenger substances such as MEcPP, PAP, H₂O₂, or Ca. 2+ -signals. The nucleus receives these signals and responds with a global transcriptional adaptation, including the activation of genes for hormone biosynthesis (e.g., auxin, cytokinin, abscisic acid), transport proteins, stress response factors, and plastid translation components.

[0025] This initial reaction leads to a local accumulation of hormones, which are transported via plasmodesmata, as well as via xylem and phloem, to adjacent tissues. There, the hormones induce secondary translational activation (TOR-dependent), thereby also bringing cells not directly treated into the same growth state.

[0026] The resulting signal amplification occurs via three coupled feedback mechanisms:

[0027] • Hormone-induced nuclear reprogramming in neighboring cells, which convert their own chloroplasts into the same metabolic state,

[0028] • Plasmodesmatic transfer of plastid metabolites, ROS and regulatory RNAs,

[0029] • Systemic Acquired Signalling (SAS) via methylated hormones, ROS or Ca 2+ -waves that extend the response to distant plant organs.

[0030] This feedback cascade leads to a stable establishment of a new systemic redox and hormone balance, which is maintained for days or weeks, even if the applied mRNA has already been degraded.

[0031] Experimental confirmation:

[0032] In one embodiment, the exogenous phytoinitiator mRNA is introduced into physiologically active plant cells by microinjection. The mRNA is suspended in an aqueous buffer solution with physiological ionic conditions (10 mM MgCl2, 100 mM KCl, pH 7.5) and injected into the cytoplasm near the chloroplasts under a microinjection pressure of 40-60 hPa.

[0033] The injection volume is approximately 0.2–0.5 pL per cell, thus preserving the cell membrane. Following injection, the mRNA passively diffuses into the chloroplast stroma via stromule-like membrane protrusions or temporary pores.

[0034] Fluorescence-labeled control mRNAs (e.g., Cy3 or FAM labeling at the 5' end) showed distinct colocalization with chlorophyll autofluorescence (A = 680 nm) within 15-30 min after injection.

[0035] Translation in plastid ribosomes was confirmed by polysome analysis (sucrose gradient 0.4 M-2.0 M) and by detection of fluorescently labeled peptide products.

[0036] Determination of the dissociation constants (Kd values):

[0037] To further characterize the interaction between the applied phytoinitiator mRNAs and the plastid 70S ribosomes, dissociation constants (Kd values) were determined using fluorescence anisotropy and filter binding assays.

[0038] The measured Kd values ​​were in the range of 10' 8 up to 10' 9 mol L -1and demonstrate a high specific affinity of phytoinitiator mRNAs for ribosomal protein complexes. These values ​​differ significantly from the Kd values ​​of endogenous chloroplast mRNAs (typically 10⁻⁵). 7 mol L -1 ) and explain the preferred ribosome binding and translation of the applied mRNAs.

[0039] The measured binding strength correlates directly with the efficiency of protein biosynthesis and serves as quantitative proof of the mechanism of action of the invention.

[0040] The principle of "ribosomal competition optimization" is thus defined experimentally via Kd measurements:

[0041] The phytoinitiator mRNAs exhibit such a high ribosome affinity that even a low local concentration is sufficient to shift the plastid translation flow into a new equilibrium state without displacing the endogenous mRNA population. These results demonstrate the functional import pathway of free mRNA molecules into chloroplasts and their translation by plastid 70S ribosomes under physiological conditions.

[0042] The competitive dynamics are illustrated in the following phases:

[0043] 1. Ribosomal binding: The phytoinitiator mRNA competes with native mRNAs for ribosome binding, but its higher binding affinity ensures that it dominates the ribosomal resources (Fig. 5).

[0044] 2. Ribosomal binding vs. translation efficiency: The diagram shows how both phytoinitiator mRNA and native mRNAs compete for ribosomal resources in the chloroplast (Fig. 6).

[0045] 3. Efficiency of translation: With increasing ribosome competition, the phytoinitiator mRNA maintains its efficiency, leading to a steady increase in protein synthesis for growth-promoting functions (Fig. 7).

[0046] 4. Feedback regulation: Over time, the ribosomes adapt to the mRNA with higher affinity, resulting in sustained protein synthesis even when the native mRNA concentration increases (Fig. 8).

[0047] B. Ribosome dynamics and mRNA competition

[0048] The phytoinitiator mRNA is structured to outperform native mRNA by maintaining high-affinity binding to chloroplast ribosomes, thereby maximizing the availability of the translation machinery for growth-related protein production. Ribosome competition between native mRNAs and phytoinitiator mRNA ensures that key proteins are produced more efficiently, leading to improved growth under various environmental conditions (Fig. 1).

[0049] In addition to ribosomal competition within chloroplasts, the invention also encompasses the translation of exogenous mRNAs at cytosolic ribosomes. These proteins can be imported into chloroplasts after their synthesis, where they modulate signaling processes.

[0050] The invention thus covers two complementary mechanisms - direct translation in the chloroplast and translation in the cytosol with subsequent protein import.

[0051] C. Ribosome Availability as a Parameter for Measuring Efficiency In the chloroplast, the efficiency of translation is closely linked to how many ribosomes are free and available to interact with the mRNA, thereby controlling the rate of protein synthesis. Ribosome availability reflects how efficiently the mRNA can bind to the ribosomes, which is a crucial step in translation.

[0052] The translation potential of chloroplasts depends on the binding of ribosomes to mRNA to produce proteins, and the availability of ribosomes (along with other translation factors) determines how efficiently this occurs.

[0053] Using ribosome availability as a measure:

[0054] - Higher ribosome availability means that more ribosomes are ready to initiate translation, resulting in faster or more efficient protein synthesis.

[0055] - The limited availability of ribosomes slows down translation, as fewer ribosomes are available to bind to the mRNA.

[0056] Ribosome availability can be used to model how quickly translation efficiency reaches saturation when ribosomes bind to mRNAs (both native and phytoinitiator mRNA). This illustrates how phytoinitiator mRNA increases translation efficiency by binding more ribosomes, leading to higher protein synthesis.

[0057] In chloroplasts, where ribosome binding represents a bottleneck for translation efficiency, ribosome availability captures the realistic, limiting factor of protein synthesis.

[0058] This approach also sheds light on how competition between mRNAs for ribosome binding influences protein synthesis. Phytoinitiator mRNAs with higher binding affinity dominate this competition, which in turn leads to increased protein production. Competition for ribosomes: Illustration of ribosome availability as a parameter for measuring efficiency. The effects of phytoinitiator mRNA on chloroplast translation efficiency based on ribosome availability are shown (Fig. 2).

[0059] Considerations: Competition for ribosomes

[0060] Phytoinitiator mRNAs have a higher binding affinity to ribosomes compared to regular mRNAs. Consequently, they compete with regular mRNAs for available ribosomes.

[0061] • Limited ribosome pool: Ribosomes are a finite resource in the chloroplast. At any given time, only a certain number of ribosomes are available to bind to mRNA and initiate translation.

[0062] • Higher affinity: Phytoinitiator mRNAs are designed to bind more efficiently to ribosomes, i.e., they have a greater chance of "catching" ribosomes than normal mRNAs.

[0063] • Dominance in translation: As a result, the phytoinitiator mRNAs dominate the available ribosomes, leading to increased protein synthesis for the specific proteins encoded by these mRNAs.

[0064] In this case, phytoinitiator mRNAs receive more ribosomes at the expense of native mRNAs, meaning that proteins encoded by native mRNAs exhibit lower synthesis than those encoded by phytoinitiator mRNAs. Alternative explanation: Increased ribosome production due to high demand. This is a more complex scenario. In some cellular environments, an increased demand for protein synthesis can stimulate the production of additional ribosomes. However, in the context of chloroplasts, this process is regulated differently than in the nucleus or cytoplasm. This scenario could work as follows:

[0065] • High demand for translation: When there is a high demand for translation

[0066] (e.g., due to the introduction of highly efficient phytoinitiator mRNAs), the chloroplast could attempt to increase the production of ribosomes to meet the demand.

[0067] • Ribosome biogenesis: Ribosomes are synthesized in response to cellular signals, but this process does not occur instantaneously. It involves the synthesis of ribosomal RNA and cellular RNA into proteins, which are then assembled into functional ribosomes. In chloroplasts, this process is highly energy-intensive, tightly regulated, and tied to the requirements of photosynthesis and other metabolic processes.

[0068] • Limited ribosome production: Unlike in the cytoplasm, where ribosomes are constantly produced according to cellular needs, ribosome biogenesis in the chloroplast is subject to greater limitations. The number of additional ribosomes the chloroplast can produce is limited because it is part of a larger regulatory system that includes both the chloroplast and nuclear genomes.

[0069] In this scenario, while additional ribosomes could be produced in response to demand, it is unlikely that the chloroplast could rapidly generate a significant surplus of ribosomes to meet the immediate high demand from the phytoinitiator mRNA. The immediate effect would still be competition for the available ribosomes.

[0070] Conclusion - Competition for ribosomes

[0071] The primary mechanism by which phytoinitiator mRNAs enhance protein synthesis is that, due to their higher binding affinity, they displace regular mRNAs for available ribosomes. While it is possible that the chloroplast might respond by producing more ribosomes over time, this process would be slow and tightly regulated. This means that the immediate effect of phytoinitiator mRNAs is due to their advantage in ribosome binding, rather than a sudden increase in ribosome production. In the short term, this competition favors phytoinitiator mRNAs, allowing them to capture more ribosomes and boost protein synthesis. Over time, the chloroplast might increase ribosome production, but this would be a secondary effect rather than the primary driver of increased translation efficiency.

[0072] 2. Results of the field trials

[0073] Extensive tests were conducted on a range of plants, including tomatoes, sugarcane, squash, and cucumbers, in both controlled and natural environments. The following results were observed for plants treated with phytoinitiators compared to untreated controls:

[0074] • Root growth: An increase in root mass of 15-30% was observed, which improves nutrient uptake and anchoring of the plant.

[0075] • Shoot development: The number of shoots increased by 10-30%, which increased plant productivity.

[0076] • Hypocotyl growth: The treated plants showed an increase in hypocotyl diameter of 5-20%, which contributes to greater structural integrity.

[0077] (Fig. 3)

[0078] It was demonstrated that the growth enhancement occurs both with translation of phytoinitiator mRNAs in chloroplasts and with translation in the cytosol followed by protein import. In both cases, the same effects result: an increase in root biomass, an increase in shoot number, and a thickening of the hypocotyl.

[0079] The invention is explained in more detail using examples with reference to the accompanying drawing.

[0080] It shows:

[0081] Fig. 1 a diagram “Ribosome dynamics and mRNA competition: phytoinitiator versus native mRNA”, Fig. 2 a diagram “Effects of phytoinitiator mRNA on chloroplast translation efficiency based on ribosome availability”,

[0082] Fig. 3 is a bar chart to illustrate the results of field trials,

[0083] Fig. 4 a diagram “Improved protein synthesis by phytoinitiator mRNA over time”,

[0084] Fig. 5 a diagram “Ribosomal binding saturation in chloroplasts based on ribosome availability”,

[0085] Fig. 6 shows a diagram “Ribosomal binding versus translation efficiency in chloroplasts”

[0086] Fig. 7 a diagram “Translation efficiency of the chloroplast based on ribosome availability and mRNA binding affinity” and

[0087] Fig. 8 shows a diagram of “competitive advantages in chloroplasts over time (based on translation efficiency)”.

[0088] In Fig. 1, the Y-axis represents ribosome occupancy and translation efficiency, and the X-axis represents time (an arbitrary unit). The higher efficiency of phytoinitiator mRNA is shown by curve 1 compared to natural mRNA, as shown by curve 2.

[0089] In Fig. 2, the Y-axis represents the efficiency of protein synthesis (translation) and the X-axis represents ribosome availability (arbitrary unit). Here, curve 1 shows: phytoinitiator mRNA Kd = 15 nM; curve 2: phytoinitiator mRNA Kd = 25 nM; curve 3: native mRNA Kd = 40 nM

[0090] Curve 4: native mRNA Kd = 45 nM

[0091] In Fig. 3, the Y-axis denotes the growth rate [%]. The two bars A1 and A2 of group A represent the increase in root mass, with bar A1 showing minimum growth in % and bar A2 showing maximum growth in %. The two bars B1 and B2 of group B represent the increase in the number of shoots, with bar B1 showing minimum growth in % and bar B2 showing maximum growth in %. The two bars C1 and C2 of group C represent the increase in hypocotyl growth, with bar C1 showing minimum growth in % and bar C2 showing maximum growth in %.

[0092] In Fig. 4, the Y-axis represents protein synthesis (arbitrary unit) and the X-axis represents time (arbitrary unit).

[0093] This graph, as shown in Fig. 4, illustrates the increased protein synthesis over time with phytoinitiator mRNA. The data demonstrate the cumulative protein synthesis and highlight how phytoinitiator mRNAs drive the rapid increase in protein production over time. This behavior reflects the efficient translation process that phytoinitiator mRNAs undergo due to their strong ribosome binding affinity (lower Kd values), enabling them to consistently produce proteins at a higher rate.

[0094] Key observations:

[0095] 1. Exponential increase in protein synthesis:

[0096] The graph shows a steady and exponential increase in protein synthesis over time. This curve illustrates that phytoinitiator mRNAs have a reinforcing effect on protein production over time due to their ability to attract ribosomes for translation.

[0097] Initially, the rate of protein synthesis is gradual, but as time progresses, the increase becomes more pronounced and exponential, indicating a highly efficient system in which more ribosomes bind to phytoinitiator mRNAs, resulting in cumulative protein production.

[0098] 2. Low initial synthesis followed by rapid growth:

[0099] ° In the initial phase (0 to 10), protein synthesis increases slowly. This initial phase represents the time required for ribosome binding and the initiation of translation. ° However, as more ribosomes bind and initiate translation, the protein synthesis rate accelerates, demonstrating the cumulative effect of ribosome-mRNA interactions over time. After the initial binding and translation phase, translation efficiency improves dramatically, resulting in rapid protein synthesis.

[0100] 3. Phytoinitiator mRNA efficiency:

[0101] The curve shows that phytoinitiator mRNAs achieve sustained growth in protein synthesis over time. Due to their low Kd values ​​(strong ribosome binding affinity), phytoinitiator mRNAs are able to initiate and maintain translation at a higher rate than other mRNAs. This ensures that they dominate protein production, resulting in continuous and efficient protein synthesis.

[0102] This efficiency is particularly important in phases where the plant requires rapid protein synthesis, such as during development, growth, or stress response.

[0103] Long-term effects of phytoinitiator mRNAs on protein synthesis:

[0104] 1. Sustainable protein production:

[0105] Over time, the phytoinitiator mRNAs lead to sustained high protein production. This is beneficial for maintaining chloroplast function, as proteins involved in photosynthesis, stress responses, and growth are produced more efficiently.

[0106] ° In the long term, the chloroplast can continuously produce important proteins, thus ensuring that the plant is constantly supplied with the components necessary for its metabolism.

[0107] 2. Rapid Growth and Stress Adaptation: The ability to ramp up protein synthesis over time gives plants with phytoinitiator mRNAs an advantage during periods of rapid growth or when adapting to stress. The exponential increase in protein production ensures that the plant can respond quickly to environmental changes such as increased light exposure or water stress by producing the proteins necessary for adaptation and survival. In the long term, this ability increases the plant's overall growth rate and its resilience to environmental fluctuations. Competitive Advantage: Because phytoinitiator mRNAs are able to synthesize proteins at a higher rate than other mRNAs, they give the plant a competitive advantage. This increased protein synthesis supports faster growth, better energy conversion, and a better ability to recover from stress.Over time, plants with more efficient phytoinitiator mRNAs will overtake those with less efficient mRNAs, as they can maintain their growth and adapt better to changing environmental conditions. Cumulative protein production: The exponential trend in the graph indicates that the cumulative effect of sustained ribosome binding and translation efficiency leads to a multiplicative increase in protein synthesis. This is particularly beneficial for chloroplasts, which require high rates of protein production to maintain cellular functions. This cumulative protein production ensures that important proteins are synthesized in large quantities over time to support both short-term responses and long-term growth.

[0108] Conclusion:

[0109] The diagram in Fig. 4 illustrates the long-term benefits of phytoinitiator mRNAs in boosting protein synthesis. Over time, their ability to consistently attract ribosomes and maintain high translation efficiency leads to a rapid increase in protein production. This enhanced protein synthesis gives plants greater adaptability, faster growth, and the ability to withstand environmental stress. The sustained high protein production of phytoinitiator mRNAs provides the chloroplast—and thus the plant—with a significant competitive advantage in terms of metabolism and survival.

[0110] In Fig. 5, the Y-axis represents ribosome binding (saturation) and the X-axis represents ribosome availability (arbitrary unit). Here, curve 1 shows: phytoinitiator mRNA Kd = 120 nM; curve 2 shows: phytoinitiator mRNA Kd = 140 nM

[0111] Curve 3: native mRNA Kd = 160 nM

[0112] Curve 4: native mRNA Kd = 190 nM

[0113] This diagram, shown in Fig. 5, illustrates the ribosomal binding saturation in chloroplasts for various mRNAs based on ribosome availability. The four curves represent different Kd values, reflecting the binding affinity between ribosomes and mRNAs. A lower Kd value indicates a stronger binding affinity, while a higher Kd value indicates a weaker binding.

[0114] The diagram in Fig. 5 shows that phytoinitiator mRNAs with lower Kd values ​​(120 nM and 140 nM) have a clear advantage in ribosomal binding and achieve higher saturation with increasing ribosome availability. Conversely, native mRNAs with higher Kd values ​​(160 nM and 190 nM) show weaker ribosomal binding saturation, indicating weaker ribosome-mRNA interactions.

[0115] Key observations:

[0116] 1. Strong binding for phytoinitiator mRNAs:

[0117] The phytoinitiator mRNAs with Kd = 120 nM and Kd = 140 nM consistently show higher ribosomal binding saturation at all levels of ribosome availability.

[0118] This result is to be expected, since a lower Kd value indicates a stronger binding affinity. These mRNAs are more likely to bind to available ribosomes, even if ribosome availability is limited.

[0119] 2. Weaker binding for native mRNAs:

[0120] The native mRNAs with Kd = 160 nM and Kd = 190 nM exhibit weaker ribosome binding, with lower saturation values.

[0121] These higher Kd values ​​correspond to a weaker binding affinity, which means that fewer ribosomes bind to these mRNAs, especially when ribosome availability is low.

[0122] 3. Gradual increase in satiety:

[0123] ° With increasing ribosome availability, all mRNAs show a gradual increase in ribosomal binding, with the phytoinitiator mRNAs retaining a distinct advantage.

[0124] ° The distance between phytoinitiator and native mRNAs remains almost constant even with high ribosome availability, suggesting that phytoinitiator mRNAs continue to dominate protein synthesis.

[0125] Long-term consequences of phytoinitiator mRNAs in chloroplasts:

[0126] 4. Dominance in Protein Synthesis: Due to their higher ribosomal binding saturation, phytoinitiator mRNAs dominate protein synthesis in the chloroplast. This leads to the chloroplast producing more proteins encoded by these mRNAs, particularly those related to growth, stress adaptation, or photosynthesis. Over time, this dominance improves chloroplast function and increases protein production, resulting in enhanced plant growth and greater adaptability. Effective Translation: The stronger ribosome binding affinity of phytoinitiator mRNAs allows the chloroplast to prioritize their translation, leading to more efficient protein production. Native mRNAs can compete for ribosome binding, reducing their translation and limiting the production of the proteins they encode.This selective translation is a potential regulatory mechanism that ensures the more effective synthesis of proteins crucial for plant growth and stress response. Increased sensitivity in response to environmental changes: The higher ribosomal binding saturation of phytoinitiator mRNAs provides the chloroplast with a mechanism for rapidly adapting protein synthesis in response to environmental factors such as light, temperature, nutrients, and stress conditions. By producing a greater number of proteins encoded by the phytoinitiator mRNAs, the chloroplast can precisely tune its function to meet the plant's metabolic needs, particularly during developmental stages such as seedling growth or adaptation to environmental stress. 7. Resource allocation:

[0127] Since ribosomes in the chloroplast are a finite resource, the higher ribosome binding saturation of phytoinitiator mRNAs means that more resources are made available for the translation of these mRNAs.

[0128] This reallocation of resources can restrict the translation of native mRNAs and prioritize proteins necessary for growth and stress adaptation. In the long term, this can lead to chloroplasts favoring the production of certain proteins over others, thus affecting the overall metabolic balance of the plant.

[0129] Conclusion:

[0130] In summary, the diagram shows that phytoinitiator mRNAs consistently outperform native mRNAs in ribosome binding within the chloroplast and maintain higher saturation levels as ribosome availability increases. This competitive advantage in ribosome binding enables phytoinitiator mRNAs to achieve higher protein synthesis rates, enhance chloroplast function, and allow the plant to adapt more flexibly to environmental changes. Over time, this can lead to selective translation, prioritized resource allocation, and improved chloroplast performance under a variety of conditions.

[0131] In Fig. 6, the Y-axis represents saturation (binding and efficiency) and the X-axis represents ribosome availability (arbitrary unit). Here, curve 1 shows: phytoinitiator mRNA Kd = 120 nM (binding); curve 2: phytoinitiator mRNA Kd = 140 nM (binding); curve 3: native mRNA Kd = 160 nM (binding); curve 4: native mRNA Kd = 190 nM (binding).

[0132] Curve 5: Phytoinitiator mRNA Kd = 120 nM (efficiency) Curve 6: Phytoinitiator mRNA Kd = 140 nM (efficiency) Curve 7: Native mRNA Kd = 160 nM (efficiency)

[0133] Curve 8: native mRNA Kd = 190 nM (efficiency)

[0134] This diagram shows the relationship between ribosomal binding saturation and translation efficiency for four different mRNAs in the chloroplast, based on ribosome availability. Each curve shows how strongly ribosomes bind to the mRNAs and how efficiently they translate the mRNAs into proteins.

[0135] Key observations:

[0136] 1. Stronger ribosome binding for phytoinitiator mRNAs:

[0137] The phytoinitiator mRNAs with lower Kd values ​​(120 nM and 140 nM) consistently exhibit stronger ribosomal binding. These mRNAs achieve higher binding saturation with the same ribosome availability compared to the native mRNAs.

[0138] A lower Kd value reflects a higher binding affinity, meaning that the phytoinitiator mRNAs displace the native mRNAs at ribosome binding.

[0139] 2. Noticeable differences between commitment and efficiency:

[0140] ° The efficiency curves for each mRNA deviate significantly from its binding saturation, suggesting that the actual translation efficiency does not correspond to the binding saturation despite strong ribosome binding.

[0141] Phytoinitiator mRNA with Kd = 120 nM has strong ribosome binding (68%), but shows a slightly lower translation efficiency (58%).

[0142] ° For the phytoinitiator mRNA with Kd = 140 nM, the binding saturation also reaches 64%, while the translation efficiency is 52%.

[0143] 3. Weaker binding and efficiency in native mRNAs: ° Native mRNAs (with Kd values ​​of 160 nM and 190 nM) exhibit weaker ribosome binding (53% and 48% respectively) and also lower translation efficiency (43% and 37% respectively).

[0144] This suggests that native mRNAs are not only less competitive at ribosome binding, but are also translated less efficiently, even when they do bind to ribosomes.

[0145] Long-term consequences of phytoinitiator mRNAs:

[0146] 1. Higher protein synthesis rates:

[0147] Phytoinitiator mRNAs dominate protein synthesis due to their stronger ribosome binding and higher translation efficiency. Over time, this leads to more efficient protein production in the chloroplast, which benefits plant growth and adaptability.

[0148] 2. Selective translation:

[0149] The chloroplast prioritizes the translation of phytoinitiator mRNAs, leading to the selective production of proteins that support growth, stress response, and other important functions. Native mRNAs may be displaced, resulting in a lower translation rate for the proteins they encode.

[0150] 3. Resource allocation:

[0151] Ribosomes are a limited resource, and the strong affinity for phytoinitiator mRNAs ensures that these mRNAs are allocated more resources. This can lead to a shift in energy and resource allocation within the chloroplast, favoring the production of proteins that are most critical for immediate needs, such as during recovery from stress or rapid growth phases.

[0152] Conclusion: In this simulation, phytoinitiator mRNAs have a clear advantage over native mRNAs in both ribosome binding and translation efficiency. While ribosome binding saturation plays an important role in determining how many ribosomes are occupied, translation efficiency is crucial for actual protein production. This diagram illustrates how competitive advantages in binding and efficiency lead to a shift in protein production, allowing the plant to respond flexibly to environmental changes and growth requirements.

[0153] In Fig. 7, the Y-axis represents translation efficiency and the X-axis represents ribosome availability (arbitrary unit). Here, curve 1 shows: phytoinitiator mRNA Kd = 120 nM; curve 2: phytoinitiator mRNA Kd = 140 nM; curve 3: native mRNA Kd = 160 nM

[0154] Curve 4: native mRNA Kd = 190 nM

[0155] This diagram, following Fig. 7, provides insight into the translation efficiency of phytoinitiator and native mRNAs, based on ribosome availability and mRNA binding affinity in the chloroplast. The four curves correspond to different Kd values, reflecting the binding strength between ribosomes and mRNAs. A lower Kd value indicates a stronger binding affinity, allowing the mRNAs to attract ribosomes for translation more effectively.

[0156] Key observations:

[0157] 1. Phytoinitiator mRNAs dominate translation efficiency:

[0158] The phytoinitiator mRNAs with Kd = 120 nM and Kd = 140 nM achieve a higher translation efficiency compared to the native mRNAs.

[0159] The phytoinitiator mRNA with Kd = 120 nM achieves a translation efficiency of 65%, while the phytoinitiator mRNA with Kd = 140 nM achieves 62%.

[0160] 2. Native mRNAs have lower translation efficiency: ° The native mRNAs with Kd = 160 nM and Kd = 190 nM have lower translation efficiency, reaching 51% and 46% respectively.

[0161] ° This lower efficiency is to be expected, since the higher Kd values ​​indicate a lower binding affinity to ribosomes, which means that fewer ribosomes are available for protein translation.

[0162] 3. Saturation of translation efficiency:

[0163] ° For all mRNA types, translation efficiency increases with increasing ribosome availability, but the difference between phytoinitiator and native mRNAs remains almost constant.

[0164] ° As ribosome availability increases, the mRNAs approach their respective efficiency limits, showing that even under optimal ribosome conditions, the phytoinitiator mRNAs are more efficient.

[0165] Long-term effects of phytoinitiator mRNAs:

[0166] 1. Improved protein synthesis:

[0167] The higher translation efficiency of phytoinitiator mRNAs leads to the chloroplast producing more proteins that are crucial for growth and stress adaptation. This increased protein production can improve chloroplast function, particularly during critical phases such as seedling development or response to environmental stress.

[0168] 2. Selective translation:

[0169] Phytoinitiator mRNAs maintain a competitive advantage in ribosome binding and translation, leading to selective protein production. This dominance can favor the synthesis of proteins involved in chloroplast stability, photosynthesis, or growth regulation. Over time, the reduced translation efficiency of native mRNAs means that certain proteins are downgraded, potentially altering the balance of protein production within the chloroplast.

[0170] 3. Flexibility and adaptability:

[0171] Phytoinitiator mRNAs provide the chloroplast with a mechanism to adapt to changing conditions by enabling rapid and efficient protein synthesis. This flexibility allows plants to respond more effectively to environmental stimuli such as light, water, or nutrient availability, thus improving their overall resilience and growth.

[0172] 4. Resource allocation and energy costs:

[0173] Since ribosomes are a limited resource, the preferential translation of phytoinitiator mRNAs can lead to a shift in resource allocation. This redistribution ensures that the chloroplast concentrates its energy on the production of proteins encoded by phytoinitiator mRNAs, which are often essential for plant growth or adaptation.

[0174] However, the plant may need to balance this increased protein production with energy constraints. If the energy demand for sustained high translation efficiency becomes too great, the chloroplast can regulate ribosome availability or mRNA levels to prevent metabolic exhaustion.

[0175] Conclusion:

[0176] In summary, this diagram illustrates the competitive advantage of phytoinitiator mRNAs with respect to translation efficiency in chloroplasts. The lower Kd values ​​of phytoinitiator mRNAs allow them to outperform native mRNAs in ribosome binding, leading to higher protein production and thus improved chloroplast function. In the long term, this dominance can lead to selective translation and influence how the chloroplast allocates its resources to maintain optimal performance and adapt to environmental changes. In Fig. 8, the Y-axis represents feedback regulation (translation efficiency) and the X-axis represents time (an arbitrary unit). Here, curve 1 shows: Phytoinitiator mRNA Kd = 120 nM; curve 2: Phytoinitiator mRNA Kd = 140 nM; curve 3: native mRNA Kd = 160 nM; curve 4: native mRNA Kd = 190 nM

[0177] The results of the diagram in Fig. 8 illustrate the competitive advantage of phytoinitiator mRNAs over native mRNAs in the chloroplast based on their translation efficiency and ribosome binding affinity. The Kd values ​​for the phytoinitiator mRNAs are lower (120 nM and 140 nM) compared to the native mRNAs (160 nM and 190 nM). A lower Kd value indicates a stronger binding affinity to ribosomes, allowing phytoinitiator mRNAs to initiate and maintain protein synthesis more efficiently.

[0178] For phytoinitiator mRNAs, this has the following consequences:

[0179] • You will achieve a higher level of efficiency in translation more quickly.

[0180] • They can maintain a higher steady-state efficiency compared to native mRNAs.

[0181] • They are less likely to be displaced by native mRNAs, even over time.

[0182] In this representation according to Fig. 8:

[0183] • Phytoinitiator mRNA with Kd = 120 nM achieves the highest efficiency of approximately 55%, thus demonstrating its strong competitive advantage.

[0184] • Phytoinitiator mRNA with Kd = 140 nM achieves an efficiency of 52%, still surpassing native mRNAs.

[0185] • Native mRNAs with Kd = 160 nM and 190 nM achieve efficiencies of 38% and 37%, respectively, demonstrating their lower translation efficiency compared to phytoinitiator mRNAs. Long-term consequences of phytoinitiator mRNAs in the chloroplast:

[0186] 1. Higher protein synthesis rates:

[0187] The consistently higher translation efficiency of phytoinitiator mRNAs leads to increased protein production over time. Since chloroplasts are responsible for the production of many important proteins, especially those involved in photosynthesis, the advantage of phytoinitiator mRNAs can lead to a significant increase in the production of these important proteins.

[0188] This higher protein synthesis rate can improve the overall function of the chloroplasts, which increases the energy efficiency and metabolic performance of the plant.

[0189] 2. Improved adaptability and growth:

[0190] Phytoinitiator mRNAs offer a distinct advantage in scenarios where the plant requires rapid growth or stress adaptation. Their ability to efficiently synthesize proteins gives the plant an edge in responding to environmental changes such as light availability, nutrient fluctuations, or water stress.

[0191] Plants with a greater capacity for efficient translation of phytoinitiator mRNAs can grow faster and produce more biomass, which is particularly advantageous during periods of rapid growth or developmental stages such as seed germination.

[0192] 3. Maintenance of chloroplast function:

[0193] Chloroplasts are essential for plant energy production, and their ability to continuously produce critical proteins is crucial for chloroplast stability. The superior translation efficiency of phytoinitiator mRNAs helps maintain this stability by ensuring a constant supply of necessary proteins, thus supporting optimal chloroplast function. This continuous protein production contributes to the long-term health and viability of chloroplasts, which are essential for the plant's energy system.

[0194] 4. Potential regulation of native mRNA translation:

[0195] Over time, the dominance of phytoinitiator mRNAs in translation can lead to regulation or even suppression of the translation of native mRNAs. Since ribosomes are a limited resource, the strong binding affinity of phytoinitiator mRNAs can limit the chances of native mRNAs being translated into proteins.

[0196] This can lead to selective protein production, in which phytoinitiator mRNAs drive the synthesis of specific proteins, possibly favoring those proteins that are crucial during growth phases or recovery from stress.

[0197] 5. Energy costs and balance:

[0198] While phytoinitiator mRNAs offer a clear advantage in protein synthesis, their high translation efficiency can come at a metabolic cost. The plant must carefully balance the energy expenditure required for a sustained high level of protein synthesis with the benefits of increased protein production.

[0199] If the energy demand to maintain this high efficiency becomes too high in the long term, the plant may need to modulate the mRNA activity of the phytoinitiator to prevent metabolic exhaustion. This can occur through feedback regulatory mechanisms that adjust mRNA levels or ribosome availability.

[0200] Conclusion: In the long term, phytoinitiator mRNAs offer a significant competitive advantage in protein synthesis in chloroplasts due to their strong ribosome binding affinity and high translation efficiency. This leads to increased protein production, improved chloroplast function, optimized plant growth, and greater adaptability. However, long-term consequences include the need to balance protein synthesis demands with the plant's energy budget and the potential regulation of native mRNA translation to ensure overall cellular homeostasis.

[0201] The invention thus encompasses a broad mechanistic spectrum: (i) direct translation of the phytoinitiator mRNAs in the chloroplasts and (ii) translation in the cytosol with subsequent protein import into the chloroplasts. Both mechanisms lead to increased protein synthesis, activation of hormonal signaling pathways, and sustained growth enhancement.

Claims

Patent claims 1. Method for improving plant growth, comprising: administration of exogenous mRNA, obtained from chloroplasts of germinating seeds, into a plant cell, wherein the mRNA is translated by ribosomes of the plant cell to produce proteins that modulate growth-related signaling pathways.

2. Method according to claim 1, characterized in that the ribosomes are chloroplast ribosomes and the mRNA translation leads directly to the synthesis of growth-promoting proteins within the chloroplast.

3. Method according to claim 1, characterized in that the ribosomes are cytosolic ribosomes and the generated proteins are imported into the chloroplasts to initiate or enhance signaling pathways for growth enhancement.

4. Method according to one of claims 1 to 3, characterized in that the application of the mRNAs leads to an increase in root biomass by 15-30%, the number of shoots by 10-30% or hypocotyl growth by 5-20% compared to untreated plants.

5. Method according to any one of claims 1 to 4, characterized in that the mRNAs are administered via leaf sprays, injections into the plant tissue or soil formulations.

6. System for improving plant growth, including: - a formulation with exogenous mRNAs derived from seedling chloroplasts and - a mechanism for introducing these mRNAs into plant cells, whereby the mRNAs are translated in the cytosol or in chloroplasts and the resulting proteins enter the chloroplasts to trigger signaling pathways for growth enhancement.

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