Overall RGR and DT clonal comparison
Independently from the gravity treatments, growth characteristics of Wolffia globosa clones analysed in terms of the relative growth rate (RGR), revealed that the seven evaluated clones reproduced not at the same rate. The one-way ANOVA results showed highly significant differences in RGR (F = 44.316; P < 0.001). Specifically, clone 9498 exhibited the highest mean RGR (0.326), while clone 9582 displayed the lowest mean RGR (0.219). These findings were further substantiated by the Student-Newman-Keuls post-hoc tests, which identified several statistically distinct groups among the clones, thereby confirming the significant inter-clone variances in growth rates (Fig. 3).
Regarding the Doubling Time (DT), the one-way ANOVA confirmed a genetic effect on the growth rate. Among the clones, the slowest (Clone 9582) took an average of three days (3.189 days) to double the surface occupied by the plants, whereas the fastest clone (9498) reached the DT in 2.151 days only, consistent with its faster growth rate (Fig. 4).
Among clones Effect of Gravity Conditions on Relative Growth Rate (RGR)Overall, the Relative Growth Rate (RGR) of Wolffia globosa clones under varying gravitational conditions showed different responses among gravity treatments (CO-RPM vs RPM and CO vs 4g). More specifically, in the CO-RPM vs RPM setting (Fig. 5a), three of the seven clones showed a significant decrease in RGR as an effect of the simulated microgravity. In more detail, clone 8356 displayed a significant alteration (F(1, 22) = 17.035, p < .001), and clone 9006 followed suit with a notable shift in RGR (F(1, 22) = 19.002, p < .001), and clone 9498 showed the least significant response (F(1, 22) = 5.565, p = .028) in the simulated microgravity versus control setting. The growth rate of the other clones resulted unaffected by the altered gravity conditions.
Turning to the effect of hypergravity (Fig. 5b), the scenario is different to what was previously observed under simulated microgravity. Although less than half of the sample showed significant differences, two showed an increase in RGR (8356 and 9910), while one showed a reduction in RGR (9582). To go in a more detailed observation of the results of the ANOVA, the most pronounced difference in this scenario was observed in Clone 9910, which showed a substantial increase in RGR (F(1, 22) = 19.446, p < .001) followed by clone 8356 (F(1, 22) = 4.459, p = .046). Lastly, clone 9582 demonstrated a marked decreased response (F(1, 22) = 7.300, p = .013).
Figure 5 –Observed mean RGR in CO-RPM vs RPM and 1g CO vs 4g. a) shows the RGR displayed by different clones under simulated microgravity (RPM), against their control (CO-RPM). b) shows the RGR displayed by different clones under hypergravity (4g), against their control (CO). Data refer to means ± SD of the means (N = 12). Statistical differences are marked by *if p < 0.05, ** if p < 0.01, *** if p < 0.001 and n.s. (not significant) if p > 0.05.
Morphological
The univariate ANOVA conducted on morphological data from Wolffia globosa under the two distinct gravitational scenarios has provided insightful information about the plant’s morphological adaptations to different gravities.
In simulated microgravity, independently from the single clones, the overall model demonstrated significant differences in the plant’s long side ratio between altered gravity and control treatments (F(13, 336) = 14.650, p < .001), underlining a base variability of the morphological traits of the plants. The results of the ANOVA showed a lower R-squared value of 0.362 compared to the hypergravity scenario of 0.418. This indicates that the variation of the Calculated Long Side Ratio among the two different gravity levels underlines a different morphological response to different gravity levels. The higher ratios observed under hypergravity conditions lead to a morphological shift towards less rounded plants. Under simulated microgravity, the Calculated Long Side Ratio was the highest in Clone 9299 (1.448), followed by Clone 9498 (1.437), while Clone 8356 exhibited the lowest ratio (1.219). Likewise, in hypergravity, the Calculated Long Side Ratio of Clone 9299 exhibited the highest ratio (1.479), followed by lone 9498 (1.445), and Clone 9006 with the lowest ratio (1.227).
In-depth analysis of the morphological reaction of single clones versus their controls under the two gravitational settings revealed that under the CO-RPM vs RPM scenario (Fig. 6a), the responses among the tested clones were generally uniform, with no significant morphological changes observed in the Calculated Long Side Ratio. This consistency suggests a general morphological stability of Wolffia globosa clones under simulated microgravity conditions.
In contrast, the CO vs 4g condition (Fig. 6b) revealed significant morphological changes in three of the seven clones. Worth noticing is that the significant differences were always towards a higher long-side/short side ratio. Clone 8356 showed the most significant difference to its control (= 18.899, p < .001), followed by clone 9006 (= 8.504, p = 0.005) and lastly, Clone KJA 0025 ( F = 5.492,p = 0.023).
Figure 6 –Observed mean long side ratio in CO-RPM vs RPM and CO vs 4g. a) shows the Long side ratio displayed by different clones under simulated microgravity (RPM), against their control (CO-RPM). b) shows the Long side ratio displayed by different clones under hypergravity (4g), against their control (CO). Data refer to means ± SD of the means (N = 25).. Statistical differences are marked by *if p < 0.05, ** if p < 0.01, *** if p < 0.001 and n.s. (not significant) if p > 0.05.
Correlation
The study explored the connection between Wolffia globosa's growth rate and its biometrical traits y. Specifically, it investigated how the plant's average growth rate (RGR Mean) relates to the average length of its longest side among the clones (as depicted in Fig. 7). The data showed a notably strong inverse link between these factors. This was evidenced by a Pearson correlation coefficient of -0.751, paired with a highly significant p-value (less than .001), indicating a robust inverse relationship. Essentially, as the average growth rate of the plant increases, the average length of the clones tends to decrease. This pattern was further validated by Spearman's rho, which yielded a correlation coefficient of -0.812 (with a p-value less than .001), reaffirming the reliability and strength of this negative correlation. These insights shed light on the fundamental growth behavior of Wolffia globosa, emphasizing the inverse relationship between its growth rate and physical size.
Protein content
In this study, the effects of varying gravitational conditions on the protein content of different clones of Wolffia globosa were analysed. Comparison between control (CO-RPM) conditions and simulated microgravity (RPM) show a significant reduction in protein content (F(1, 40) = 19.562, p < .001).
Conversely, the overall comparison between control (1g) and 4g conditions yielded no significant differences in protein content (F(1, 40) = .002, p = .962). Results showed that the increase to 4g did not markedly alter the protein synthesis or accumulation in the samples and the negligible variance explained by the model (R Squared = .000, Adjusted R Squared = − .025) further underscores the minimal impact of this gravity alteration on protein content.
Clone vs Treatments
To underline the variability in protein content between clones under simulated microgravity, we compared samples from the Control-Microgravity (CO-RPM) vs simulated microgravity (RPM) condition (Fig. 7a). Results highlighted a significant variation in response among different clones. In general, a reduction in protein content was underlined among all clones. The highest p-value was observed for clones 9299 (p = 0.001), followed by clones 9498 (p = 0.005), 9006 (p = 0.039) and KJA 0025 (p = 0.032).
Under the Control (CO) vs 4g condition (Fig. 7b), results revealed a notable reduction in protein content only in clone 9498 (p = .027), suggesting that this clone is susceptible to both altered gravity conditions in terms of protein content.
Figure 8 –Percentage of the protein content of the fresh weight (FW) in CO-RPM vs RPM and CO vs 4g. a) shows the % protein content of the FW displayed by different clones under simulated microgravity (RPM), against their control (CO-RPM). b) shows the % protein content of the FW displayed by different clones under hypergravity (4g), against their control (CO). Data refer to means ± SD of the means (N = 3). Statistical differences are marked by *if p < 0.05, ** if p < 0.01, *** if p < 0.001 and n.s. (not significant) if p > 0.05.