## 4 Discussion  
### Legume-Specific miRNAs in Gleditsia sinensis  
In this investigation, 57 legume-specific miRNAs were found in _G. sinensis_, which provides further molecular proof of the molecular classification of _G. sinensis_ as Leguminosae plants. Among nine miRNAs with differential expression in ponds during different developmental stages, the targets of _miR2658_, _miR4414b_, _miR4415a-3p_, _miR4994-3p_, _miR8213-5p_, and _miR8736_ were ranked in the top 20 GO or KEGG enrichment pathways (Table S5, Figures S3 and S4). Interestingly, the target annotation showed that many of these miRNAs were involved in stress responses. For example, _miR5213-5p_ was induced from May to July, and its targets were predicted to regulate phytochemicalamin biosynthetic/metabolic processes in response to heavy-metal stress [34] and plant-pathogen interaction pathways. The result was similar to that of previous reports in that _miR85213-5p_ could regulate abiotic stress by targeting NBS-LRR genes [35]. In plant-pathogen interaction pathways, it was shown that other targets of _miR8213-5p_ belong to genes encoding NBS-LRR-family proteins produced to resist pathogenic stress [36], implying that _miR8213-5p_ might negatively regulate the response to pathogen infection. Similarly, _miR4414b_ was also suggested to be related to the plant-pathogen interaction pathway. Additionally, some of the remaining legume-specific miRNAs with no differential expression in _G. sinensis_ might also be involved in abiotic or biotic stress responses. For example, _miR85368_ and _miR8512_ were found to respond to drought stress in alfalfa [35]. Through degradome sequencing, _miR8748_ was validated as targeting genes encoding HSP90 (heat-shock protein) and the transcription factor _MYC_2 to respond to selenium hyperaccumulation [37]. _miR1507a_ was reported to be involved in the resistance of soybeans to pathogen infection by regulating several NBS-LRR-family disease resistance genes during ETI [38]. However, the function of some species-specific miRNAs in this investigation was not annotated due to the incomplete genome of _G. sinensis_; nevertheless, the regulatory roles of these miRNAs were reported in other plants. For example, _miR85559_ was shown to respond to water stress in Caragana korshinskii in the Loes Plateau [39] and could regulate chilling injury [40].  
The overexpression of _gma-miR8150a/b_ could reduce resistance to Phytophthorahora sojae by suppressing the expression of NBS-LRR genes [41]. Thus far, in Leguminosae, the molecular mechanism of these legume-specific miRNAs, which are involved in the response to multiple stresses for adaption for better growth, could not be explained, especially in _G. sinensis_, a perennial woody plant.  
Intriguingly, another molecular proof that supports _G. sinensis_ being classified as a legume is the existence of the miRNAs in _G. sinensis_ related to symbiotic process, which exist widely in _Leguminosae. miR4416_ was reported to regulate the nodule number by targeting _GmRIPT_ (rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene) [42]. In addition, thus far there are no reports that nitrogen-fixing rhizobium exists in _G. sinensis_; however, there might be other beneficial symbiotic bacteria for better survival, adaption, and growth of _G. sinensis_, which could be supported by the annotation that one of the targets of _miR84414b_ in _G. sinensis_ was suggested to be involved in intracellular protein transport in a symbiotic interaction.  
Moreover, we found that _miR862b_, _miR85671a_, _miR85037c_, _miR2089-3p_, and _miR1520n_ might only be conserved in legumes, but other members in these families could be found in non-legume species [43; 44; 45; 46; 47].