Structure-Property-Function Relationship in Humic Substances to Explain the Biological Activity in Plants

Knowledge of the structure-property-function relationship of humic substances (HSs) is key for understanding their role in soil. Despite progress, studies on this topic are still under discussion. We analyzed 37 humic fractions with respect to their isotopic composition, structural characteristics, and properties responsible for stimulating plant root parameters. We showed that regardless of the source of origin of the carbon (C3 or C4), soil-extracted HSs and humic acids (HAs) are structurally similar to each other. The more labile and functionalized HS fraction is responsible for root emission, whereas the more recalcitrant and less functionalized HA fraction is related to root growth. Labile structures promote root stimulation at lower concentrations, while recalcitrant structures require higher concentrations to promote a similar stimulus. These findings show that lability and recalcitrance, which are derived properties of humic fractions, are related to the type and intensity of their bioactivity. In summary, the comparison of humic fractions allowed a better understanding of the relationship between the source of origin of plant carbon and the structure, properties, and type and intensity of the bioactivity of HSs in plants. In this study, scientific concepts are unified and the basis for the agronomic use of HSs is established.

. 13 C CP MAS NMR spectra of the different humic fractions extracted from organic soils and composted materials.  Structural characteristics of humic fractions analyzed by FTIR. The absorption bands at ~3400 cm -1 indicate the presence of stretching vibrations () -OH and/or -NH (alcohols, carboxylic acids and amides). The bands at ~2900 cm -1 and 2800 cm -1 correspond to symmetric and asymmetric -CH, respectively (-CH 3 aliphatic). An intense band stands out in the HS fractions at ~1593 cm -1 corresponding to C=C vibrations of aromatic structures and symmetric and asymmetric -COO -. In the HA fractions, the absorption band at ~1715 cm -1 assigned to  -COOH of carboxylic acids was more visible. Visible bands between 1617 cm -1 and 1620 cm -1 in the Hu and HA fractions, respectively, are complex bands corresponding to  C=C aromatic,  C=O of amide I, and symmetric  -COO -(in the HA the asymmetric  -COOwas visible at 1407 cm -1 ). Complex bands at 1388 cm -1 and 1378 cm -1 visible in HSs and HAs correspond to  -OH, -CH 2 and -CH 3 deformations and  -CO of phenols. The bands at ~1035 cm -1 and ~1097 cm -1 correspond to  -OH of aliphatic alcohols and polysaccharides.     In this additional study, the humic fractions of the soils from the State of Rio de Janeiro (humic substances (HS): HS_RJ, humic acids (HA): HA_RJ and fulvic acids (FA): FA_RJ), from the State of Rio Grande do Norte (HS_RN, HA_RN and FA_RN) and from the State of Mato Grosso do Sul, Brazil (HS_MS, HA_MS and FA_MS) were used. Additionally, the humic fractions from the vermicompost (VCF) (HS_VCF, HA_VCF and FA_VCF) were also used.
The FA fractions were extracted according to the procedure described by the IHSS 5 . In general, the liquid humic fraction that remains after centrifugation of the precipitated humic acids was passed through a column loaded with the resin Supelite TM DAX-8, Cod. 21567-U (commercial substitute of Sigma for XAD-8). Next, the fulvic acids retained in the column were eluted with an NaOH solution (0.1 M), and the column was later washed with distilled water for total recovery of the fulvic acids. The fulvic acids were then passed through an ion exchange column (Amberlite IR120 hydrogen form, Fluka analytical Cod. 06428), and the solution was lyophilized.
The structural characterization of the FA was performed as described in the Materials and Methods section of the manuscript.
The bioactivity experiments were carried out under the same conditions as those described in the Materials and Methods section of the manuscript.
The root parameters were evaluated as described in the Materials and Methods section of the manuscript.
The structure-activity relationship was established as described in the Materials and Methods section of the manuscript.

Results and Discussion
13 C NMR and PCA characterization of the spectral data from the different humic fractions.
As expected, the soil-extracted FAs were shown to have structural characteristics independent of the HA and HS fractions. However, the data confirm our research hypothesis and reaffirm the results reported in the manuscript because the HA and HS fractions of the VCF are structurally similar to the whole HS fractions extracted from the soils. In the PCA-13 C NMR (71% of the total variance explained), the negative values on PC1 (55%) indicate that the FA extracted from the VC are structurally similar to the whole HS fractions (Fig. S7). This same behavior was observed when comparing the soil HA and HS fractions between the compost and vermicompost using PCA (Figure 1-manuscript).

Biological Activity
The FA fractions were shown to have biological activity, stimulating plant root growth; these results have already been observed in the literature 6,7 . The FA concentrations (2.5-5.0 mg (C) .L -1 ) with the greatest stimulation of root growth were lower than those for the HAs (5.0-10.0 mg (C) .L -1 ). Thus, the FAs showed a range of maximum effect similar to that shown by the HS (Fig. S8). The PCA (90% of the total variance explained) that relates the humic fractions to the root parameters is shown in Fig. S9A. The plot shows that both the FA fractions as well as the whole HSs (positive values on PC1-80%) exert effects on the smaller roots and on the number of smaller roots. These results can be interpreted as effects on the emission as well as the length and surface area of the roots. The HAs were shown to exert effects on the diameter of the roots and larger roots, which can be interpreted as effects that are more related to root growth and vigor. The PCA (90% of the total variance explained) that relates these effects with the structures is shown in Fig. S9B. The HS and FA fractions showed a relationship (positive values on PC1-80%) between predominantly more functionalized structures (C-substituted) and their effects on root emission, in contrast to the HAs, which showed a relationship with negative values on PC1 for structures that were predominantly less functionalized (C-unsubstituted) and root growth and diameter parameters. These results again confirm the findings of the studies shown in the manuscript and prove that the most functionalized structures could be responsible for the emission of roots within the mode of action of the HSs on plants, while the less functionalized structures could exert more direct effects on root growth and thickening (Fig. S9).

Conclusions
The results presented in these new experiments are in agreement with the conclusions of the main manuscript. The lability, a property resulting from the structural characteristics and obtained through a statistical tool, is shown again as a viable parameter for the interpretation of the relationships between the structure, property and functions of the HSs, and which was also confirmed in this case for the FAs. Additionally, this experiment suggests the existence of a supramolecular structural organization of the HSs as a whole. Our results indicate that the hydrophobic and hydrophilic domains of the HSs show a type of specific action in the regulation of root growth. Our results are coherent with those published by Canellas 8 , where it was proven that the hydrophobic domain of the HSs can be broken to release the hydrophilic structures responsible for the stimulation and emission of roots in plants.