*3.2. LiNACs Are Closely Associated with the Weeping Trait of L. indica*

It has been suggested that the weeping trait may be associated with abnormal GA signaling [31]. The drooping phenomenon also occurs when the gravity response changes [32]. Changes in the distribution of plant hormones often affect the formation of plant xylem and bast, which is another factor contributing to the phenomenon of weeping plants. Lignin is a phenylpropanoid-derived polymer that, together with cellulose and hemicellulose, forms the cell wall of plant vascular tissue and provides mechanical support for upright plant growth [33,34]. The NAC family of transcription factors is the master switch in the regulation of secondary cell wall thickening in plants and influences the plant phenotype by regulating the growth of secondary cell walls in plants. The cell wall thickness of upright branches in *P. mume* was significantly higher than that of weeping branches. In terms of overall expression, *PmWND1*(*PmNAC082*) [35] regulated downstream secondary-wallsynthesis-related genes, which were all significantly less expressed in the weeping branches than in the upright branches, indicating that *NAC* may play an important role in regulating the production of weeping traits in plants.

In this study, qRT-PCR analysis of upright *L. indica* 'Shaoguifei' and weeping *L. indica* 'Xiariwuniang' at three different growth stages showed that *LiNACs* played an important role in the weeping trait of *L. indica* and different *LiNACs* expression patterns were different during this process. The expression of *LiNAC13*, a member of the NAC1 subgroup, did not change significantly in the three growth stages of 'Shaoguifei', but decreased gradually in 'Xiariwuniang' and was much lower than that of 'Shaoguifei'. The expression of *LiNAC13* in 'Xiariwuniang' decreased gradually and was much lower than that of 'Shaoguifei'. NAC1 is normally involved in hormone signaling in plants to regulate plant growth and development [26] and it is speculated that *LiNAC13* may be involved in regulating weeping traits in plants by participating in the phytohormone signaling pathway, but this remains to be verified. The relative expression of *LiNAC2* in S1–S3 is always higher in 'Xiariwuniang' than in 'Shaoguifei', especially in the S1–S2 stages, and there is not much difference between them in the S3 stage. *LiNAC2* is closely related to AT3G105001/*NTL4*, so we hypothesized that *LiNAC2* began to negatively regulate the weeping trait of *L. indica* at S1. This still needs to be further verified by subsequent experiments. *LiNAC12* is closely related to AT5G13180.1 (*VNI2*), a VNI2 transcription factor that plays a molecular linkage role between plant response to environmental stress and regulation of leaf longevity [36]. In contrast, the expression of *LiNAC12* in 'Xiariwuniang' was always higher than that of 'Shaoguifei' and reached a peak during S2. It is speculated that 'Xiariwuniang' may have an advantage over 'Shaoguifei' in regulating plant growth and coping with environmental stress during development, which may also be a reason for its weeping branch formation. The relative expression of *LiNAC8* (which is closely related to AT1G32770.1/*SND1*) gradually increased in the three growth stages (S1–S3) of 'Shaoguifei' and 'Xiariwuniang' branches and the expression of 'Shaoguifei' was always higher than that of 'Xiariwuniang'. In our study, by measuring the lignin content in 'Shaoguifei' and 'Xiariwuniang' branches at different growth stages, we found that the lignin content in 'Shaoguifei' was always higher than that of 'Xiariwuniang'. Further, in the early stage of growth, there was not much difference in the content between the two, but with the development, especially at the end of the growth stage, the lignin content in 'Shaoguifei' was much higher than that of 'Xiariwuniang' (Figure 6). Inhibition of *SND1* expression in *Arabidopsis* leads to a dramatic decrease in the

expression of all three secondary wall synthesis genes and pedicel fiber cells are unable to form secondary walls and droop grow [15]. In addition, dominant inhibition of *PtrWNDs* in poplar caused significant thinning of secondary walls of transgenic poplar stem fibers and failure of plants to grow upright [18]. Lignin is an important component in the secondary cell wall of plants and when the lignin content increases, the fruit of *Eriobotrya japonica* also becomes hard [21]. Therefore, we speculate that 'Shaoguifei' has more lignin content than 'Xiariwuniang' and the branches of 'Shaoguifei' provide more mechanical support, while 'Xiariwuniang' has less lignin content and, therefore, droops. *SND5* in *Arabidopsis* is closely connected to *SND2/3*, which is involved with the secondary wall and its direct homolog in poplar, according to Zhong et al. [19]. Similar findings were found in our study, *LiNAC8* showed a consistent trend with lignin content at S1–S3 in different *L. indica* species, which indicated that *LiNAC8* may be involved in affecting the synthesis of secondary walls and determining the weeping trait of *L. indica*. However, this conclusion has to be further investigated.
