**5. Conclusions**

The importance of GABA for human health and plant development has been confirmed. In this study, high GABA levels were detected in seeds before germination, implying GABA not only could influence chestnut seed germination, but also could act as a potential compound of functional chestnut food. This should be examined in greater detail in future studies. The application of exogenous GABA and VGB inhibited chestnut seed germination and early primary root growth, possibly by altering the balance between carbon and nitrogen metabolism, especially the free amino acid contents before germination (Figure 8). The data presented herein suggested that changes to the endogenous GABA levels in chestnut seeds might adversely a ffect germination. This insight may be relevant for improving the storage of chestnut and other recalcitrant seeds over winter.

**Figure 8.** Model of the effects of exogenous GABA and VGB on primary carbon and nitrogen metabolism during chestnut seed germination at 48 and 120 h. Red: increase; green: decrease; black: no significant change; G: GABA vs. CK; V: VGB vs. CK.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/9/4/449/s1, Table S1: Data of PCA analysis of exogenous GABA and VGB on the physiological effects during chestnut seed germination.

**Author Contributions:** Manuscript draft: S.S., C.D., and W.C.; experiment: C.D. and W.C.; analysis: C.D., W.C., J.S., G.W., Y.W., J.Z., J.J., and D.Y.; conception and supervision: S.S. and Z.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from the Project of Science and Technology Development Center of State Forestry and grassland administration (KJZXSA2019045); the National Natural Science Foundation of China (31971627); the Project of Key Laboratory of Tree Breeding and Cultivation of State Forestry and Grassland Administration (ZDRIF201712); the Science & Technology Research and Development Program of Hebei Province (16226312D).

**Acknowledgments:** We thank Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
