To the best of our knowledge, this study was the first to demonstrate how in situ plants generate seeds by adapting to variable nutrient environments, in terms of the metabolome of developing seeds in subtropical regions, where P was limiting for plant growth and development. Based on our results, the discussions focused on differences in the metabolic profiles of acorns, and the identification of metabolites that played key roles in metabolic regulation at the contrasting P sites.
Sequential dynamic in C- and N-containing metabolites during acorn development
Our results clearly revealed that, for the oak populations at the two sites of contrasting P availability, from July to September, concentrations of most sugars, amino acids, and organic acids in acorns initially increased and then decreased (Additional file 4: Table S1; Figures 2, Additional file 2: Figure S2), with PI values of the same metabolomic pathways in the three developmental stages different (Additional file 5: Table S2). These results suggested that, during developing, metabolic states of acorns changed based on the sequentially dynamic of C- and N- containing metabolites. This was consistent with the results of Wang et al. , who found that the metabolites and proteins involved in the development of Lotus (Nelumbo nucifera) seeds were sequentially dynamic. On one hand, this might be caused by the vital function of these metabolites in seed development. From July to August, acorns synthesized many C- and N- containing metabolites (e.g., amino acids, sugars, and organic acids) to synthesize the storage substances in September, when acorns reached the physiological maturation with the dry mass being relatively stable (Figure 1) . As reported by others, key metabolites (e.g., amino acids, sugars, and organic acids) were significantly altered in developing maize , lotus , and wheat , even in developing fruits, such as, medlar (Mespilus germanica) , navel oranges (Citrus sinensis) , and ponkan (Citrus reticulata) .
On the other hand, the sequentially dynamic of metabolites in developing acorns at the contrasting-P sites might be related to the ordered regulation of genes in metabolite synthesis during plant development. The significant reduction of C- and N- containing metabolites was regulated by gene expression . Although the most active genes in seeds were shared throughout development, each developmental stage utilized a set of genes that was up-regulated compared with other stages, or special at the GeneChip level . Similarly, the transcriptome research of developing acorns by Miguel et al.  revealed that, carbohydrate metabolism, including sugar metabolism, the TCA cycle, and amino acid metabolism, was most represented during acorn development, but every stage had special up-regulated metabolisms, such as carbohydrates and energy metabolism for the middle stage. Similarly, even though some special metabolisms appeared in July, sugar metabolism, the TCA cycle, and amino acid metabolism comprised the core metabolic pathways in developing acorns (Figure 2; Additional file 5: Table S2), with the size and dry mass increasing significantly from July to September at both P-rich and P-deficient sites (p < 0.05) (Figure 1).
Besides, as a storage organ, acorns primarily accumulated starch (46.3% - 68.6%), followed by fats and proteins . Therefore, during acorn development at the two contrasting-P sites, noticeable changes occurred in the concentrations of sugars, sugar alcohols, and amino acids, and the changes were related to their functions in the synthesis of starch and proteins (Figure 2). Firstly, as a form of energy for plants, starch steadily accumulated during acorn development , resulting in the remarkable increase of acorn dry mass (Figure 1). During this process, small sugar molecules (particularly sucrose and d-fructose) were converted to large sugar molecules (e.g., raffinose and starch) [38, 41], coupled with the consumption of sugar alcohols (e.g., myo-inositol and galactitol) (Figure 2) , or acted as vital regulators [51, 52]. The accumulated large sugar molecules would serve as energy reserves (Figure 2) , which could provide energy materials in the establishment of seedlings. Hence, at the two sites, during maturation process (from August to September), concentrations of sucrose and d-fructose, as well as myo-inositol and galactitol decreased significantly (Figures 3, Additional file 2: Figure S2; Additional file 4: Table S1). Secondly, amino acids, which were generally synthesized in leaves, could be transported into seeds for further protein synthesis , and acted as intermediates for glycolysis and the TCA cycle in energy metabolism (Figure 2) [55-57]. Hence, the decrease of most amino acids (particularly l-glutamic acid) during maturation (Additional file 2: Figure S2; Additional file 4: Table S1) might have been caused by the incorporation of amino acids in the synthesis of storage proteins.
Differential C- and N-containing metabolites in oak acorn populations at P-rich and P-deficient sites
Our results clearly showed that metabolites, predominantly sugars and organic acids, differed significantly in acorns of oak populations at the P-rich and P-deficient sites (Figure 5). This suggested that these types of sugars and organic acids were involved in the responses of in situ plant seeds to the variable supplies of soil nutrients due to geologically-derived environments. This had been proved by Ji et al. , who found that sugars and organic acids of Q. variabilis leaves played predominant roles in clearly discriminating Q. variabilis trees at the contrasting geologic-P sites. The vital functions of sugars and organic acids were also showed in other studies on the response of plant leaves and seeds under different nutritional conditions. Yan et al.  revealed that the added P could increase concentrations of sugars in oilseed flax seeds. P deficiencies  and Zn stress  predominantly altered concentrations of sugars and organic acids in tea (Camellia sinensis) leaves.
As the reproductive organ of Q. variabilis trees, acorns were characterized by the enrichment of starch during the development process . Carbohydrate metabolism, including glycolysis, gluconeogenesis, amino sugar and nucleotide sugar metabolism, served as the essential biochemical process (Figure 2) . As two vital forms of metabolites, sugars and organic acids hold a larger percentage in plants than other metabolites, and sugars (e.g., fructose, sucrose, glucose) and organic acids (e.g., shikimate, glycine, glutamate) were the primary intermediates that participated in the carbon and nitrogen metabolism of plant seeds (Figure 2) . Similarly, the important functions of sugars and organic acids were observed in developing fruits, such as ponkan (Citrus reticulata) , navel oranges (Citrus sinensis) , and medlar (Mespilus germanica) .
Increased efficiency of P use in the late stage acorns at P-deficient sites over P-rich sites
In September, the dry mass of Q. variabilis acorns reached stable (Figure 1). We found that there were distinct metabolic shifts in acorns of this stage at such contrasting geologic-P sites (Figure 6). The distinct metabolic shift of plants under different nutrient supply situation has been reported in other studies. Christian and Oliver  showed that Chlamydomonas reinhardtii developed highly distinctive metabolite profiles under N, P, S, or Fe deficiency conditions. Gargallogarriga et al.  revealed that increased P availability could lead to shifts in the metabolome through higher investments in the protection mechanisms of plants.
Concretely, our results indicated that the utilization of P was different to guarantee the same dry mass in acorns at P-rich and P-deficient sites with different soil nutrient supply (Figure 6). Erythrose could be synthesized by erythrose-4-phosphate, which was an important intermediate product of the pentose phosphate pathway [63, 64], and the pentose phosphate pathway consumed less inorganic P than glycolysis . In our study, the concentrations of most sugars, most organic acids, orthophosphoric acid, and P were lower, and erythrose was higher in the late-stage acorns at P-deficient sites than those at P-rich sites. But the dry mass and C concentration showed no significant differences in late-stage acorns at the two sites (Figure 6; Additional file 6: Table S3). Unlikely, seeds of some Proteaceae species were considered to accumulate comparatively higher P concentration in seeds to adapt to low P environments , and produce small seeds in such P-impoverished habitats . P addition could enhance the dry mass of maize (Zea mays) . Besides, shifts in P usage were associated with variable P supplies to plants [69-71]. Hence, late-stage acorns at P-deficient sites might boost the pentose phosphate pathway to decrease P quotas through bypassing P consumption in glycolysis reactions, which increased the efficiency of P use in acorns at P-deficient sites (Figure 6).
Further, similar to previous reports concerning the element concentrations of other organs [25, 72], the protein/nucleic-related (e.g., N, P, and S) and enzyme-related (e.g., Mn, Cu) elements of late-stage acorns were more highly impacted, in contrast to other types of elements at P-rich and P-deficient sites in the present study (Figure 6). However, unlike our results, supply of P could enhance the concentrations of macro-elements (e.g., N, P, and S) in crops, such as urdbean (Vigna mungo) , wheat (Triticum aestivum), maize (Zea mays), and faba bean (Vicia faba) , but decrease the concentrations of micro-elements (e.g., Mn and Cu) in rice (Oryza sativa) . Considering the strongly close relationships between these elements (e.g., N, P, S, Mn, and Cu) and metabolites in plants (Figure 6) , and the vital effects of these elements on the synthesis of storage substances in plant seeds and the yield of plant seeds (Figure 6) [75, 77, 78], it also could prove the distinct metabolic shifts in the late-stage acorns at the two sites.