*2.2. Dark-Induced Senescence in Hwaseong and CR2002*

To determine whether *qCC2* is associated with senescence under dark condition, detached leaves of Hwaseong and CR2002 were incubated on 3 mM MES buffer at 27 ◦C under complete darkness (Figure 2). Hwaseong leaves turned yellow four days after incubation (DAI), while CR2002 remained green (Figure 2a). Although the chlorophyll content of CR2002 was higher than Hwaseong at 0 DAI, a larger difference was observed at 4 DAI between CR2002 and Hwaseong (Figure 2b). The efficiency of photosystem II (*Fv*/*Fm* ratio) of CR2002 was significantly higher than Hwaseong, indicating CR2002 showed delayed senescence in the DIS assay (Figure 2c).

**Figure 2.** CR2002 showed delayed senescence under dark-induced senescence (DIS) conditions. Hwaseong and CR2002 were grown in a paddy field, and fully expanded flag leaves at the heading stage were used for DIS. (**a**) Detached leaves were incubated in 3 mM MES buffer (pH 5.8) at 27 ◦C under dark conditions. (**b**,**c**) Total chlorophyll contents (*n*= 6) and *Fv*/*Fm* ratio (*n*= 5) were compared between Hwaseong and CR2002. Error bars indicate a standard error, and more than three samples were used for each experiment. \* and \*\*\* indicate significant difference at *p* < 0.05 and *p* < 0.001 based on Student's *t*-test, respectively.

To further examine the different senescence of Hwaseong and CR2002, transcript levels of SAGs (*OsNAP*, *Osh36,* and *OsI57*) and CDGs (*SGR*, *RCCR1*, *PAO*, *NYC1*, and *NOL*) were measured by qRT-PCR using detached leaf samples (Figure 3). During DIS, transcription levels of the SAGs and CDGs showed increases of 4.9-fold to 325-fold in Hwaseong, whereas increases of 4.7-fold to 200-fold were observed in CR2002. Transcript levels of SAGs and CDGs in CR2002 were significantly lower than Hwaseong at 4 DAI, with the exception of *OsI57*, indicating that the lower expression of SAGs and CDGs in CR2002 might result in the delayed senescence exhibited by CR2002 in the DIS assay.

**Figure 3.** Expression of senescence-associated genes and chlorophyll degradation genes in Hwaseong and CR2002. qRT-PCR was conducted to determine the transcript level of genes. *OsUBQ5* was used for normalization. Error bars indicate the standard deviation of three replications. \*, \*\*, and \*\*\* indicate significant difference at *p* < 0.05, *p* < 0.01, and *p* < 0.001 based on Student's *t*-test, respectively. DAI: days after incubation.
