**1. Introduction**

Chlorophyll (Chl) is a photosynthetic pigment that is an essential component of the plant photosystem. It changes solar energy to chemical energy. Because of its photosynthetic ability, increasing Chl content in crops may be an effective way to increase grain yield and biomass production [1]. A number of studies have demonstrated that Chl content is controlled by quantitative trait locus (QTL) in various genetic backgrounds in rice [2–6].

Senescence or biological aging is the final stage of plant development [7]. During senescence, leaf color turns from green to yellow because of chlorophyll degradation [8]. Leaf yellowing is frequently used as a senescence indicator. Stay-green (non-yellowing) mutants maintain leaf greenness after the grain-ripening stage [9]. Stay-green mutants exhibit delayed leaf senescence and have been found in various plant species [8,10]. In rice, several stay-green genes have been cloned and characterized, including *Stay-Green Rice* (*SGR*), *NON-YELLOW COLORING 1* (*NYC1*), *NYC1-LIKE* (*NOL*), *Oryza sativa NAC-like, activated by Apetala3*/*Pistillata* (*OsNAP*) [10–14]. *SGR,* a highly conserved senescence-associated gene, encodes a novel chloroplast protein, and the expression of *SGR* is up-regulated in both natural and dark-induced senescence. This gene interacts with the light-harvesting chlorophyll-binding protein (LHCP) [10,14]. *NYC1* and *NOL* encode short-chain dehydrogenase/reductase (SDR) [11,12]. These two genes are co-localized in the thylakoid membrane and function as a chlorophyll *b* reductase. *OsNAP* contains a typical NAC structure at the N terminus [13]. This gene plays a role in regulating leaf senescence and acts as a key component, linking ABA signaling in rice. These genes have been used as ideal markers for the onset of the senescence process.

Ubiquitination is the post-translational modification of protein substrates [15]. The ubiquitin-proteasome pathway is known to play an important role in plant seed and organ size determination, such as *DA1*, *DA2*, *EOD1*/*BB*, *SAMBA*, and *SOD*/*UBP15* genes in *Arabidopsis* [16–20]. A major function of E3 ubiquitin ligase is to regulate polyubiquitination and to degrade their target substrate proteins. E3 ubiquitin ligase *BigBrother* (*BB*) controls final organ size and seed size acting in parallel with the *DA1* gene in *Arabidopsis,* and these two genes also positively control leaf senescence [17,19,20]. Delayed senescence has been confirmed by measuring the expression of *AHK3*, *CRF6*, and *ARF2*. The negative regulators of senescence—*AHK3* and *CRF6*—are expressed higher in *da1-1\_bb*/*eod1-2* leaves, while the auxin repressor *ARF2* is expressed at a lower level than wild type [19]. *DA2,* an ortholog of *OsGW2*, controls seed and organ size by interacting with *DA1* [18].

*OsGW2*, a QTL on chromosome 2, controls grain width and weight in rice and encodes RING-type E3 ubiquitin ligase [21]. This gene might function in the degradation by the ubiquitin-proteasome pathway, and loss-of-function in *OsGW2* increases cell numbers and grain size [21]. The *OsGW2* mutant also shows increased transcript levels of *OsPCR1* during the developing grain stage, leading to an increase in Zn concentration in the seed [22]. Yeast two-hybrid and in vitro pull-down assays have shown that OsGW2 directly interacts with expansin-like 1 (EXPLA1), chitinase 14 (CHT14), and phosphoglycerate kinase (PGK) [23,24]. Transcription activation activity has been found in the *GW2*-C terminus (205 to 260) [24]. Together, the findings that OsGW2 interacts with various proteins and that the mutation of *OsGW2* has a pleiotropic effect in rice raises the possibility that *GW2* is also involved in regulating leaf senescence.

The objective of this study was to identify QTL controlling chlorophyll content and leaf senescence and to characterize candidate genes associated with this trait. *qCC2*, a major QTL for chlorophyll content, was mapped using an introgression line, CR2002, developed from an interspecific cross between *O. sativa cv*. Hwaseong and the wild species *O. grandiglumis.* CR2002 harboring *qCC2* from *O. grandiglumis* showed delayed leaf yellowing and a higher *Fv*/*Fm* value than Hwaseong. Endogenous expression levels of senescence-associated genes (SAGs) and chlorophyll degradation genes (CDGs) in CR2002 were lower than in Hwaseong. To determine if *GW2* is allelic to *qCC2* and thereby associated with delayed senescence, a *gw2*-knockout mutant (hereafter termed *gw2-ko*) was examined using dark-induced senescence (DIS) assay. *gw2*-*ko* showed delayed leaf senescence under dark conditions with down-regulated expression of SAGs and CDGs. Delayed senescence was confirmed by segregation analysis in the F2 generation. Comparative RNA-seq analysis was conducted to identify differentially-expressed transcripts, using 30-day-old leaves from Hwaseong and CR2002 that were subjected to dark treatment. This enabled the identification of which genotype-specific expressions were enriched in CR2002 and reduced in Hwaseong and vice versa, including genes involved in phytohormone biosynthesis and signaling, NAC transcription factors, and senescence-associated genes. Collectively, the results of this study suggested that *OsGW2* controlled chlorophyll content and was a positive regulator of leaf senescence in rice.
