*3.3. Di*ff*erentially Expressed Genes Associated with Leaf Senescence*

Comparative RNA-seq analysis was conducted to identify differentially-expressed transcripts, using 30-day-old leaves from Hwaseong and CR2002 to gain a better understanding of the mechanism involved in delayed senescence. A total of 10,339 and 9885 DEGs were identified in Hwaseong and CR2002 under dark treatment compared with the control, respectively (Figure 8a). To examine the difference between Hwaseong and CR2002, Hwaseong- and CR2002-specific DEGs were extracted. In CR2002-specific DEGs, 33 GO terms were enriched, and these included iron ion transport, response to hormone stimulus, cell cycle, response to endogenous stimulus, and chloroplast. Transporter genes are closely associated with leaf senescence process. During senescence, remobilization of nutrients from senescing cells to developing tissues is mediated by transporters, and transporter genes show notable changes in expression [13,29]. Chloroplast GO term contains 15 CR2002-specific DEGs, including Os07g0462000 (*OsSG1*), Os07g0558500 (*NYC4*), Os02g0152400 (*RbcS1*) [28,30]. Os07g0462000 encodes glutamate-cysteine ligase and possibly controls chlorophyll content and stay-green phenotype [30]. Os07g0558500 (*NYC4*) is an ortholog of *THF1* in *Arabidopsis.* The *AtTHF1* is known as a multi-function protein involved in acclimation to high light, sugar sensing, and disease resistance, while *nyc4-1* mutant shows the stay-green phenotype in rice [28]. Os02g0152400 encodes the rubisco small subunit 1, which reflects involvement in the regulation of rubisco catalytic activity.

Transcription factors (TFs) play an important role in regulating leaf senescence. NAC (NAM, ATAF1, and CUC2) transcription factor family is one of the large TF families, and many NAC TFs are up-regulated during leaf senescence [31]. In addition, several NAC TFs regulating leaf senescence have been characterized in various plant species [13,32–36]. Transcript levels of NAC transcription factors (*OsNAP, ONAC120*, and *ONAC122*) were specifically up-regulated in Hwaseong (Table 3). *OsNAP* is a positive regulator of leaf senescence, and its orthologs *TtNAM-B1* and *AtNAP* have shown similar function in wheat and *Arabidopsis* [13,32,33]. *OsNAC122* (or *OsNAC10*, Os11g0126900) over-expression and root-specific expression transgenic have shown significantly improved drought, high salinity, and low-temperature tolerance in rice [37].

Phytohormone signaling and biosynthesis-related genes were identified in Hwaseong- and CR2002-specific DEGs. During dark treatment, ethylene (*OsERF5*), ABA (*OsABI1*), and jasmonic acid (*OsJAZ1*) signaling genes were significantly down-regulated, while auxin synthesis (*OsNIT2*), brassinosteroid biosynthesis (*OsDWF1*), and cytokinin signaling (*OsRR3*) genes were up-regulated in CR2002 (Table 3). In contrast, ethylene biosynthesis and signaling (*OsACS2* and *OsERF2*) and auxin signaling (*OsARF2*) genes were up-regulated in Hwaseong, whereas cytokinin signaling (*OsRR6*, *OsRR9*, and *OsRR10*), brassinosteroid signaling (*OsBRI1*), and *gibberellin 20-oxidase* (*OsGA20ox1*) were down-regulated (Table 3). These results indicated that delayed leaf senescence in CR2002 (*gw2-ko*) was associated with phytohormone signaling or biosynthesis pathways in rice. The rice stay-green mutant *oself3.1* has shown down-regulated transcript levels of ABA-, ethylene-, JA-associated genes in microarray analysis [7]. The results from the present study were consistent with the report that *OsELF3.1* promoted leaf senescence by modulating signaling pathways [7]. To further investigate this relationship with phytohormones, gene expression of *GW2* in response to plant hormones was examined using the RiceXPro database. *GW2* did not show large changes in response to plant hormones (data not shown) [38]. In durum wheat, a *GW2* knock-down mutant has shown enhanced *cytokinin dehydrogenase 1* (*CKX1*) transcript level and down-regulation of *cytokinin dehydrogenase 2* (*CKX2*) and *gibberellin oxidase 3* (*GA3-ox*) [39]. In addition, a wheat near-isogenic line (NIL) harboring the *TaGW2-6A* allelic variant has shown an increase in not only grain size but also endogenous cytokinin content (Z+ZR) [40]. Compared to the control (Chinese Spring), the expression level of cytokinin biosynthesis genes (*TaIPT2*, *TaIPT3*, *TaIPT5*, and *TaIPT8*) are found to be up-regulated, while cytokinin degradation genes (*TaCKX1*, *TaCKX2*, and *TaCKX6*) are found to be down-regulated in the NIL during the endosperm development [40]. These results supported the possibility that delayed senescence in CR2002 and *gw2-ko* was mediated by phytohormone-related pathways.

#### *3.4. GW2 as an E3 Ubiquitin Ligase*

The ubiquitination process is a post-translational modification. E3 ligases function in the regulation of polyubiquitination and are involved in target protein degradation. Several studies have reported interacting partners of GW2. Target proteins of GW2 have been revealed through yeast two-hybrid screening [24]. Yeast two-hybrid and pull-down assays have shown that expansin-like 1 (EXPLA1) directly interacts with *GW2*. In addition, in vitro assays have identified the ubiquitination of EXPLA1 by *GW2* at lysine 279 (K279). Proteomic analysis has shown that chitinase 14 (CHT14) and phosphoglycerate kinase (PGK) directly interact with GW2 [23]. However, genes that are directly associated with leaf senescence have not been reported. Identification of GW2 substrate protein(s) responsible for leaf senescence would be key to understanding the post-translational molecular mechanism of leaf senescence in rice.

### **4. Materials and Methods**
