The effect of parental origin on seed size has been recognized 9,23. This study examined several F1 hybrids that develop larger mature embryos compared with their parental lines. Hybrids of C24 and Ler-1 have been suggested to promote or suppress embryo heterosis 9. We found that the use of the Rld-1 and C24 accessions as maternal donors increased F1 seed size (Figs. 1 and S3). Thus, for some of the F1 hybrid combinations examined here, it is possible that F1-hybrid-specific modifications based on maternal lineage traits caused the increase in seed size. A possible explanation for this observation is that the embryos of F1 hybrid seeds obtained by reciprocal crosses are epigenetically different even though they are genetically identical. In addition, the composition of the seed coat is affected by the maternal origin, suggesting that the contribution from the two parents is unequal. It is also possible that the maternal-derived organelle genome (chloroplast and mitochondrial DNA), which is important for seed size heterosis, exhibited different interactions with the nuclear genome in the two parents compared with the hybrid lines 24,25.
C24 × Ler hybrids exhibit a high level of heterosis regarding biomass production in Arabidopsis26–28, with upregulation of photosynthesis-related genes and early emergence of new leaves 26. The seeds of reciprocal C24 × Ler hybrids germinated 7–12 h earlier than did their parents 27. However, when the germination times of the parental and F1 hybrids were matched, the chloroplast-related genes of F1 hybrids were not downregulated or changed compared with their parents 29, suggesting that the changes detected in C24 × Ler hybrids may be attributed to differences in developmental stages between the hybrids and their parents. This is a potential explanation for the differences observed in the developmental stages of the hybrids and their parents. Therefore, in this study, the germination times were identical among the lines to eliminate the effect of the differences in developmental stages. In addition, early germination is considered an indicator of a heterotic phenotype 27. However, in our data from multiple intraspecific hybrids, none of the nine combinations that exhibited high heterosis in seedlings showed early germination in hybrids (Figs. 1 and S4). Thus, although an early germination time may contribute to a higher biomass 26,27, it does not necessarily seem to be an indicator of heterosis in Arabidopsis.
Carbohydrates or sugars are a primary source of carbon and energy and play a fundamental role in plant growth regulation 30–32. Arabidopsis allotetraploids were reported to contain more starch and sugars, as well as an increased biomass, with almost 12% more chlorophyll and 10% more starch than the BPV 33. Therefore, we assessed whether sugar derivatives were associated with the heterotic characteristics in several F1 hybrids. In this study, the hybrids were grown under short-day conditions without sucrose added to the medium, thus limiting the carbon source required for growth. We hypothesized that if sucrose is in fact involved in the heterosis mechanism, then differences in sugar-induced carbohydrate metabolic pathways in hybrids should clearly emerge. Sucrose may play a role in the induction of faster growth rates, especially during early development. In our study, trehalose tended to be lower in all F1 hybrids compared with inbred lines under short-day conditions without sucrose (Dataset S3). However, in our study, there were no clear differences in the overall carbohydrate metabolic pathway and no changes in metabolites at different levels of heterosis. In addition, the sugar-transport-responsive STP1, STP14, ERD6, and PLT6 genes were upregulated in the C24 × Ler, C24 × Col, and Col × Ler hybrids 34. Nevertheless, our transcriptome data showed no significant upregulation of these genes in Col × C24 and C24 × Col compared with their parents (Fig. S8). These results suggest that the carbohydrate metabolite profiling is not altered when biomass heterosis occurs in the F1 hybrids of Arabidopsis under short-day growing conditions.
Regarding the close association between biomass increase and metabolic composition, it has been reported that central metabolic pathways, i.e., the TCA cycle and amino acid pathway, are negatively correlated with biomass gain 14. A QTL analysis in recombinant inbred lines (RILs) and the closely related species Col × C24 and C24 × Col revealed that FUM2 was a candidate gene for heterosis 35. Recently, associations between heterotic growth traits and fumarate and malate levels, as well as high fumarate/malate ratios, were detected in large populations of Arabidopsis accessions, RILs, near-isogenic lines, hybrids, and different photoperiods 18,22. In this study, several intraspecific hybrids were used to identify metabolites associated with growth. As a result, we observed higher fumarate/malate ratios in hybrids showing high heterosis levels, similar to that reported previously (Fig. 6). Thus, the TCA cycle appears to be a core factor associated with vigorous growth. In oxygen metabolism, the TCA cycle is essential for large energy intermediates 17. Fumaric acid has a lower pKa value than that of malic acid, and a higher fumarate/malate ratio provides an advantageous mechanism for the pH-mediated regulation of intracellular flux and biomass 36. In Asparagus sprengeri, a decrease in cytosolic pH is associated with γ-aminobutyrate (GABA) synthesis (glutamate + H+ ≥ GABA + CO2) 37. The fum2 knock-down mutant exhibited a higher malate content, whereas the fumarate content remained low, and a decrease in biomass was detected under high-nitrogen conditions. This indicates that nitrate is not a major source of ammonium and that fumarate and malate help in maintaining the pH as nitrate is converted to ammonium 36. The final core function of the TCA process is the production of energy 38. Moreover, evidence has emerged in support of higher TCA cycle fluxes, photosynthetic capacity, and crop yields afforded by stomatal opening and closing movements 39–41. At the protein level, enzymes that participate in the TCA and Calvin–Benson cycles were broadly upregulated, indicating an increase in the flux to provide more energy for enhancing growth at longer photoperiods 42. The details of the upregulation of photosynthesis by controlling fumarate and malate concentrations remain unclear 39,41. In this report, the TCA cycle may have been regulated in the parental accessions, thus limiting the energy flux necessary for growth. In the case of high heterosis, hybrids can accelerate the TCA flux and can produce more energy as biomass. Previously, reduced levels of TCA cycle intermediates were shown to result in enhanced rates of photosynthesis and aerial growth throughout carbon flux 14,43. This negative correlation seems to indicate that strong growth is triggered to diminish the levels of central metabolites, rather than an increase in the supply of the components used for cellular synthesis 14. These examples reveal the importance of mitochondrial reactions for cellular function and, hence, for a higher biomass in plants. Therefore, we hypothesized that photosynthesis and/or nitrogen metabolism occurs when heterosis has a high TCA cycle flux. Because TCA cycle-related genes do not exhibit any significant changes in their transcriptional levels, we assumed that the reduced levels observed in TCA cycle intermediates occurred at the post-transcriptional level or were caused by the action of specific regulatory factors. The detailed effects of these regulatory mechanisms involved in such interactions remain to be studied.
In conclusion, we report here that general physiological traits, such as seed area and germination time, were affected by the maternal donor. In addition, the minimization of environmental differences and interindividual variability, which affect biomass and developmental stages, led to various levels of heterosis and provided taxonomy of combinations for further study. The metabolomics analysis of intraspecific Arabidopsis thaliana revealed that TCA fluxes seem to be involved in the acquisition of heterotic phenotypes. This will provide insight into the phenomena of heterosis and higher plant growth.