**6. NYE/SGR protein role during drought-mediated chlorophyll degradation**

During senescence, plant-recycled valuable nutrient components from the leaves and leaf chlorophyll (Chl) are usually converted to a colorless product through six known Chl catab‐ olism enzymes (CCEs) and a metal-chelating substance (MCS) [70]. First, Chl *b* reductase (CBR) reduces 7-formyl group of Chl *b* to a hydroxymethyl group. There are two CBR isoforms which are encoded by *NON-YELLOW COLORING 1* (*NYC1*) and *NYC-LIKE* (*NOL*) genes. Another key regulator of Chl degradation is hydroxymethyl Chl reductase (HCAR), which reduces Chl *b* to Chl *a* [71, 72]. HCAR has recently been identified in *Arabidopsis*. Then, the Mg+ atom of Chl *a* is removed by MCS, which is then called pheophytin *a* (Phein *a*). Pheophytinase (PPH) produces Pheophorbide *a* (Pheide *a*) by catalyzing Phein *a*. Subsequently, the chlorin macro‐ cycle of Pheide *a* is oxygenolytically opened by Pheide *a* oxygenase (PAO) [73], and red Chl catabolite (RCC) which is the product of this reaction is reduced to a nonphototoxic primary fluorescent Chl catabolite (*p*FCC) by RCC reductase (RCCR). Besides CCEs and MCS, STAY-GREEN1 (SGR1) or nonyellowing mutant can cause a stay-green phenotype in many plant species, such as *Arabidopsis* and rice [74]. The *NYE1* (nonyellowing) gene in *Arabidopsis* has been identified by positional cloning. NYE1 is now widely referred as SGR (Stay GReen) [75]. The *nye1-1* mutant could retain 50% chlorophyll at the end of 6-day dark incubation, whereas the wild type (*Columbia-0*) has resulted in the degradation of chlorophyll to less than 10% [75]. In addition to that, qPCR result has outlined *AtNYE1* as an extremely induced gene by senescence signals [75].

In *Festuca arundinacea*, this gene is called NONYELLOWING PROTEIN1 [nonyellowing gene (NYE)], which has 278 amino acids [76]. *FaNYE1* or *SGR1* has a high similarity to *Arabidopsis NYE1*, either by sequences or by function. *FaNYE* has been identified recently by RACE-PCR [76]. Overexpression of *AtNYE1* results in pale-yellow leaves to even albino seedlings [74]. Degreening phenotype in tall fescue occur during severe stress conditions and harsh seasons. BLAST analysis revealed that *FaNYE* has 89% sequence similarity to *Triticum urartu* Tumanian ex Gandilyan and 83% sequence similarity to *Hordeum vulgare* (NCBI). The negative correlation of *FaNYE* transcript with chlorophyll content has been previously addressed in *F. arundina‐ cea* affected by dark treatment and natural senescence, which in 9 days' dark incubation augmented *FaNYE* transcript level just about 52-fold. Furthermore, overexpressing *FaNYE* ORF in *Col-0* background leads to accelerated senescence [76]. Leaf chlorophyll concentration of the mutant diminished very slowly in rice *sgr* mutant, but steeply decreased in wild type during the grain filling. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves [77].

We hypothesized that the SGR-mediated chlorophyll degradation in fescue is genetically dependent on *FaSGR*. To confirm this, we evaluated the *FaSGR* transcripts in our selected genotypes during drought stress by real-time PCR. The substantial increase in *FaSGR* transcript in Pixie and Mini-mustang cultivars suggests their SGR-mediated chlorophyll degradation. The *FaNYE1/SGR* role in chlorophyll degradation during dark treatment and natural senescence has been published [76]. During stress conditions, chloroplast metabolism modulates leaf senescence. Hence, prevention of chlorophyll degradation can be an interesting subject in order to generate new varieties of plants with higher performance. In a preliminary transient experiment on tall fescue leaves, we targeted tall fescue *SGR* via CRISPR-Cas9 technology under heat and salinity stress, and then realized that the leaves that were exposed to CRISPR-Cas9 construct vividly maintained more chlorophyll than control lines.
