Mechanism for enhancing the growth of mung bean seedlings under simulated microgravity

The study addresses how simulated microgravity enhances growth during the early developmental stages of plants, focusing on mung bean seedlings. Spaceflight-associated microgravity poses health risks to humans, and plants are important for bioregenerative life support; therefore, understanding plant growth in microgravity is critical for space agriculture. Ground-based devices like clinostats and random positioning machines simulate microgravity, with faster rotation (20–60 rpm) providing better averaging of gravity vectors for small samples. Prior observations indicate some species exhibit accelerated early growth and enhanced phytochemicals under real and simulated microgravity. Seed germination begins with water uptake (imbibition), regulated by aquaporins, followed by mobilization of seed reserves via enzymes such as α- and β-amylases to supply sugars for growth before photosynthesis initiates. The authors hypothesized that simulated microgravity alters water uptake and reserve mobilization (amylolysis), thereby enhancing early growth, and they aimed to elucidate the underlying mechanism in mung bean using growth measurements, aquaporin and amylase gene expression, and enzyme activity assays.

The authors summarize evidence that microgravity (spaceflight and simulations) can enhance early plant development and phytochemical accumulation: Arabidopsis in space showed longer seedlings and larger leaves; Brassica napus and mung bean under clinorotation exhibited enhanced growth; Brassica rapa and soybean seedlings accumulated more phytochemicals in space; antioxidant activity increased in mung bean under clinorotation (2 rpm) and antidiabetic properties were enhanced in wheatgrass under RPM. Fast clinorotation (20–60 rpm) is commonly used for small samples to simulate microgravity more effectively than slow rotations or RPM in some contexts. Reports suggest microgravity upregulates aquaporins (e.g., rice calli) and facilitates water transport (enhanced guttation in rice), consistent with improved water uptake. Conversely, hypergravity suppresses α-amylase in wheat, implying microgravity may promote amylase expression/activity in starchy seeds like mung bean. Despite these observations, mechanisms linking microgravity to water transport and reserve mobilization during germination remain insufficiently understood.

Experimental design: Mung bean seeds were germinated and cultivated under simulated microgravity using a clinostat (clinorotation at 20 rpm) for 3 days, with parallel 1 g controls. Eight seeds were positioned 2 cm from the rotation axis in an acrylic housing arranged in a circle. Apparatus: An AC servo motor (SGMAH-A584AA21, Yaskawa Electric, Japan) drove a cylindrical acrylic housing (9.4 cm diameter × 9.0 cm height) filled with 0.8% w/v agar medium (3.0 cm depth). Measurements: Growth parameters included fresh weight, water content, and organ lengths (shoot/stem and root). Water distribution/state was monitored via near-infrared (NIR) imaging; images used second derivative intensity at 1418 nm to assess water-related signals. Gene expression: Real-time PCR quantified aquaporin transcripts in roots (plasma membrane intrinsic proteins, PIPs; and tonoplast intrinsic proteins, TIPs) and amylase genes in cotyledons (α-amylase, α-amylase 2, β-amylase, β-amylase 1). Specific primer pairs were used with gene annotations and accession numbers provided (e.g., XM_0141695502 for α-amylase; various PIP/TIP accessions). Enzyme assay: Amylase activity in cotyledons was assayed; sample size for gene expression and enzyme assays was n = 10. Imaging and schematic: NIR images contrasted clinorotation vs control seedlings; a schematic illustrated seed placement and rotation geometry. Statistical analysis: Significance between clinorotation and control groups was indicated by asterisks in tables/figures (details of statistical tests not specified in the provided text).

• Growth enhancement under clinorotation: Fresh weight increased to 332.33 ± 8.53 mg vs 298.67 ± 6.94 mg (control); water content increased to 85.49 ± 0.21% vs 83.66 ± 0.45% (control); overall length and stem length were greater under clinorotation (e.g., length 4.43 ± 0.36 cm vs 3.35 ± 0.50 cm; stem 5.05 ± 0.25 cm vs 3.50 ± 0.31 cm). Asterisks in Table 1 indicate significant differences.
• Water distribution: NIR imaging showed no distinct change in water distribution patterns between control and clinorotation seedlings, suggesting increased water content was not due to restricted evaporation; stems exhibited higher water signal consistent with elongation.
• Aquaporin expression upregulated: In roots, several aquaporin genes showed higher expression under clinorotation (e.g., PIP-1-4: 1.54 ± 0.07*, PIP-2-1: 2.32 ± 0.10* relative expression), indicating enhanced water uptake capacity; some other PIP/TIP transcripts showed smaller or non-significant changes.
• Amylase gene expression upregulated in cotyledons (relative expression ×10^−2): α-amylase 11.24 ± 1.98* vs 3.24 ± 0.72; α-amylase 2: 6.74 ± 1.38* vs 1.81 ± 0.36; β-amylase: 4.37 ± 0.79* vs 0.70 ± 0.35; β-amylase 1: 29.15 ± 3.70* vs 9.19 ± 2.00.
• Amylase activity increased: Cotyledon amylase activity was 27% higher under clinorotation (n = 10; significant difference indicated).

The results support a mechanistic sequence in which simulated microgravity (clinorotation) enhances water uptake during germination via upregulation of aquaporin genes in roots (PIP and selected TIPs). Increased water status does not arise from reduced evaporation, as NIR imaging showed similar water distribution between treatments. Enhanced hydration appears to trigger elevated expression of starch-degrading enzymes (α- and β-amylases) in cotyledons, increasing amylase activity and promoting mobilization of seed reserves to support rapid early growth. The authors propose an energy-sparing effect under microgravity: plants need not expend energy to maintain homeostasis against a constant gravity vector, allowing surplus energy to be allocated to growth processes and possibly to aquaporin regulation. This interpretation aligns with prior observations of aquaporin upregulation in rice calli and enhanced water transport (guttation) in rice seedlings under microgravity, and with reports that hypergravity suppresses α-amylase (implying microgravity may have the opposite effect). Collectively, the findings provide a plausible mechanism linking microgravity to accelerated early growth and potential enhancement of phytochemical properties through improved water uptake and reserve mobilization.

Simulated microgravity generated by clinorotation (20 rpm) enhances early growth of mung bean seedlings by upregulating aquaporin expression in roots, increasing water content, and promoting amylase gene expression and activity in cotyledons. This coordinated response accelerates reserve mobilization and seedling elongation. The work identifies aquaporin activation as a likely initial trigger for microgravity-induced growth enhancement and suggests that plants exhibiting robust aquaporin and hydrolytic enzyme responses under microgravity are promising for space agriculture. Future research should test additional plant species, examine broader gene networks and metabolic outcomes, and validate these mechanisms under real microgravity conditions in space to assess generalizability and practical application.

• Microgravity was simulated using a clinostat; simulated microgravity is not identical to real microgravity, potentially limiting generalizability to spaceflight conditions.
• Single species (mung bean) and early developmental stage were studied; responses may be species- and stage-specific.
• Short experimental duration (3 days) and relatively small sample sizes (e.g., n = 10 for gene expression and enzyme assays) may limit detection of longer-term effects.
• Statistical methods and full methodological details (e.g., exact amylase assay conditions) are not fully described in the provided text.
• Few prior reports exist on amylase regulation under microgravity, complicating direct comparison across studies.