Greenness stands out. Its variations mirror the environment and ecological characteristics, so much so that AI models can depict landscapes. Greenness is often associated with richness. The loss of greenness in plants is a natural process in plant development and maturation, characterising senescence. The breakdown of chlorophyll during senescence, whose catabolic products are unpigmented, reveals other pigments, such as carotenoids, anthocyanins, and xanthophylls

Stay-green (sometimes staygreen) is a secondary heritable trait (governed by genes but also influenced by the environment) that enables crop plants to maintain the greenness of their leaves, thereby holding photosynthetic capacity for a longer time (Thomas and Smart 1993). Genetic factors involved in stay-green phenotypes vary, and the expression patterns of these genes influence phenotypic expression, implying their utility for crop improvement.

Stay-green plants usually delay senescence. They help to tolerate abiotic stresses such as drought or heat and resist pests and diseases. These plants produce higher yields than non-stay-green plants. The stay-green trait has been used for resistance breeding for nearly four to five decades against insect pests and diseases in maize breeding for the European corn borer (Ostrinia nubilalis) and Helminthosporium turcicum, which causes northern corn leaf blight (Duvick 1984), and for breeding durable broad-spectrum resistance in cucumber against oomycete downy mildew (Pseudoperonospora cubensis), bacterial angular leaf spot (Pseudomonas syringae pv. lachrymans), and fungal anthracnose (Colletotrichum orbicular, syn. Colletotrichum lagenarium) pathogens (Wang et al. 2019).

Various researchers have identified genes referred to by different names that govern the stay-green phenotype in bunchgrass, rice, peas, and Arabidopsis (see Fang et al. 2014). In rice, the stay-green phenotype is governed by the recessive nuclear gene sgr(t) (stay-green), identified in a glutinous japonica rice mutant of Hwacheong-wx induced by N-methyl-N-nitrosourea mutagenesis (Cha et al. 2002). The mutational effect has been linked to limiting chlorophyll degradation without affecting photosynthesis. Leaf senescence, the final stage of leaf development in cereal crops, is characteristic of nutrient remobilisation and allocation, which are important features in plant growth, maturity, and yield. Asian rice cultivars primarily belong to two ecological types: Oryza sativa ssp. japonica and indica. These two subspecies exhibit distinct morphological and physiological features and demonstrate drastically different lifespans, with indica showing early senescence (Shin et al. 2020).

Stay-green phenotypes are known to occur in a variety of crops (Thomas and Smart 1993). Fang et al. (1998), in a leaf senescence study on chlorophyll degradation, reasoned that the potential inactivity of Mg-dechelatase, an enzyme that catalyses the removal of magnesium from chlorophyll in the key step of chlorophyll degradation of the non-yellowing mutant of Phaseolus vulgaris, is significant in explaining the non-yellowing/stay-green trait. Subsequently, Shimoda et al. (2016) showed that the dominant allele, SGR (Mendel’s green cotyledon gene), encodes Mg-dechelatase. This enzyme

 

catalyses the removal of magnesium from chlorophyll during its degradation (Fig. 1). Studies on the molecular regulation of chlorophyll degradation are becoming increasingly prominent. Balancing light-dependent chlorophyll biosynthesis and its degradation is of great consequence for maintaining chlorophyll homeostasis (Fu et al. 2025). The loss-of-function SGR mutant exhibits a strong stay-green phenotype without exception (Sato et al. 2007), signifying chlorophyll retention. Even though it is known that SGR homologues influence chlorophyll degradation by integrating with chlorophyll catabolic enzymes, the regulatory mechanism of SGR remains poorly understood. Nevertheless, the stay-green trait is a target in breeding strategies for crop improvement, as it enhances crop yield, quality, and pest and disease resistance (Duvick 1984, Jiao et al. 2020).

Three independent groups of researchers recently generated mechanistic insights into the stay-green phenotype and its effect on yield from different perspectives. The first group, from a Chinese team led by Wensheng Zhao, identified a plant-specific, atypical DNA binding with one finger (Dof) (also known as a zinc finger) transcription factor named OsDes1in a japonica rice cv. Aichiasahi. OsDes1 governs the stay-green phenotype, grain yield, and multi-pathogen resistance to Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) but not in indica rice (Qiu et al. 2024 and references therein). Rice has 30 Dof genes, mostly related to flowering time, seed germination, sugar transport, and the response to abiotic stress. OsDof24 delays leaf senescence by deactivating jasmonate biosynthetic pathways (Shim et al. 2019).

Qiu et al. (2024) have also identified a type of gain-of-function dominant mutant, des1-D (Delayed Senescence 1-Dominant), which produces a stay-green phenotype in a screen of transferred DNA (T-DNA) insertion lines of Aichiasahi.

In the dominant mutant, the mutation creates a new allele that coexists with the wild-type allele and is expressed. Dominant mutants are useful for studying gene functions. Both M. oryzae and Xoo induce OsDes1 expression in des1-D mutants and OsDes1-OE (overexpressing) transgenic plants, resulting in enhanced disease resistance, whereas the OsDes1-KO (knockout line) and WT (wild-type) plants develop typical susceptibility lesions.

Mechanistically, the Dof transcription factor OsDes1 interacts with the iron-sulfur (FeS) protein OsPetC (Rieske FeS protein, named after the discoverers John S. Rieske and co-workers, alternatively known as plastohydroquinone: plastocyanin oxidoreductase iron-sulfur protein) by targeting its promoter for its activation   (Fig. 2., Qiu et al. 2024). This protects against the degradation of OsPetC and promotes the expression of the stay-green trait. Moreover, the Rieske FeS protein, the core subunit of Cytb6f (cytochrome b6f) encoded by the nuclear PetC gene, is a key component of the photosynthetic electron transport pathway, thus contributing to increased yield. OsDes1 also binds to the promoters of defence-related genes, such as OsPR1b, and activates their expression, leading to enhanced disease resistance. In line with this and the reaction to the respective pathogens, the upregulation of the pathogenesis-related protein genes OsPR1b, OsPR2, and OsPR8 in the des1-D and OsDes1-OE plants supports the basis for multi-pathogen resistance in rice. In contrast, the OsDes1-KN (knockout) and wild-type plants are susceptible to both diseases.

The second group was led by Jintao Cheng from China and Ekkehard Neuhaus from Germany (Cheng et al. 2024). This study stems from the fact that many plant species accumulate sugars in senescing leaves, and the perplexing association of sugar transporters with senescence is functionally unclear. This gap in understanding highlights the significance of the sugar transport system for an efficient source-sink relationship. Proper sugar transport can obviously delay senescence, maintain photosynthetic activity, and potentially extend the productive phase of the plant.

This study followed the molecular events of senescence in Arabidopsis leaves of the wild type and the T-DNA insertion mutant. The cytosolic sugar levels differ during developmental senescence (when wild types exhibit chlorotic senescing leaves). The association of the SFP1 (SUGAR-PORTER FAMILY PROTEIN 1) gene, belonging to the family of monosaccharide transporters, with leaf senescence is known (Quirino et al. 2001), but its functions remain unclear. Rationally, Cheng et al. (2024) focus on the sugar transporters (Fig. 3). Their study demonstrates the induction of the vacuolar SENESCENCE-ASSOCIATED SUGAR TRANSPORTER 1 (SAST1, syn: SFP1), which results in low cytosolic glucose levels due to the export of glucose to the vacuole. This low energy signal regulates two master regulators: (i) it represses TOR1 (TARGET OF RAPAMYCIN 1) activity and promotes SNF1-related protein kinase 1 (SnRK1) (SUCROSE NON-FERMENTING PROTEIN KINASE 1) expression. This results in decreased TOR activity, triggering autophagy, a natural process for breaking down unnecessary cellular products or damaged parts for recycling, involving autophagy-related genes (ATGs): ATG7, ATG8, ATG11, and ATG18, which represent the senescent state of the leaves. Thus, the ratio between TOR and SnRK1 activity is crucial for regulated leaf senescence. In contrast, in sast1 T-DNA insertion mutants, the high energy state induces the master regulator, TOR activity, concomitantly suppressing SnRK1 activity. As a result, both the autophagy genes (ATGs) and the senescence-related genes, ProDH (PROLINE DEHYDROGENASE), DIN6 (GLUTAMIN-DEPENDENT ASPARAGINE SYNTHASE 6), and SEN1 (SENESCENCE-ASSOCIATED PROTEIN 1), except for HDT1 (HISTONE DEACETYLASE 1), are expressed at lower levels. This causes a stay-green phenotype, which maintains a high anabolic state. However, despite the stay-green phenotype, the sast1 mutants produce a reduced seed yield. Nevertheless, this study points to the possibility of regulating sugar compartmentation mediated by SAST1 for proper leaf senescence, which is necessary for the stay-green phenotype to positively affect plant yield.

Still, another recent study by a third Chinese team of researchers led by Shiming Zuo identified yet another natural mutant of Stay-Green (OsSGR) in a japonica rice cultivar YanDao 8 (YD8) cultivated in the field, which confers resistance to sheath blight in rice, hence named sbr1 (sheath blight resistance 1), without affecting grain yield or quality, besides expressing the stay-green phenotype (Xie et al. 2024). In the absence of clear-cut genes governing resistance to an economically important disease like sheath blight, the potential of the sbr1 gene to confer improved resistance to this disease gains significance.

The infection of rice by the Rhizoctonia solani sheath blight pathogen induces the expression of OsSGR and upregulates genes for OsCKX (cytokinin dehydrogenase). This results in accelerated degradation of chlorophyll and cytokinins, initiating the process of leaf senescence and affecting leaf sheaths, which facilitates pathogen invasion. (Fig. 4). The sbr1/Ossgr mutant gene has a distinct 444 bp insertion in the second exon compared to the wild-type gene, leading to a truncated protein. The mutation reduces the function of most OsCKX genes, which regulate cytokinin (CK) biosynthesis. Cytokinin alters the cell cycle, cytoskeleton, and endocytosis in Botrytis cinerea, a necrotrophic fungal pathogen. It affects cytoskeletal components and cellular trafficking (Gupta et al. 2021). This results in lowered endocytic rates and reduced endomembrane compartment sizes, leading to arrested growth and limiting the fungal pathogen’s developmental programmes. In this way, the accumulation of cytokinin in the sbr1 stay-green mutant may confer resistance to sheath blight caused by the necrotrophic fungal pathogen R. solani.

However, the authors record that this stay-green mutant line is undesirable because it interferes with proper crop harvesting and field management for the succeeding crop; therefore, breeders and farmers do not want it. Nevertheless, with an understanding of the functioning of the OsCKX genes as susceptibility genes, Xie et al. (2024) engineered transgene-free sheath blight resistance by knocking out a specific OsCKX7, which is more effective at inducing sheath blight susceptibility in another commercially popular japonica cultivar, XuDao3, using CRISPR/Cas9 genome-editing technology. The phenotypic traits of these osckx7 lines are similar to those of the wild type, including the expression of regular developmental senescence (normal yellowing) while demonstrating sheath blight resistance. This inconsistency with the stay-green hypothesis underscores the differences among cytokinin dehydrogenases in maintaining chlorophyll retention, which is evident in the visual phenotypic trait.

Taken together, the naturally occurring recessive allele, sgr of the stay-green gene SGR, or alternatively, OsDes1, is a promising target for improving rice with a stay-green phenotype, grain yield, and multi-pathogen resistance. The sbr1 gene is also a valuable genetic resource for rice sheath blight resistance with a refinement of its expression. Hybrid sterility caused by reproductive isolation creates problems in transferring japonica-specific genes, such as sgr and OsDes1, to indica subspecies. However, advancements in the genetic and molecular basis of indica-japonica hybrid sterility offer new approaches for transferring genes from japonica to indica, thereby overcoming the sterility problem (Zhang 2020). Furthermore, the positive regulation of sugar compartmentation in stay-green phenotypes, highlighted by Cheng et al. (2024), opens new and challenging avenues for introducing appropriate mutations into crop species to balance stay-green attributes and crop yield. Further, the stay-green study with the sbr1 gene linked OsCKX genes, which break down cytokinin, to disease susceptibility and revealed the possibility of engineering resistance by modifying these genes, which is not related to the stay-green phenotype.

Loss of chlorophyll due to degradation during developmental senescence and its delay or retention in the stay-green phenotypes is a hallmark of this trait. The involvement of other pathways regulating the stay-green phenotype by three independent groups has generated mechanistic insights, leading to alternative genes’ utility for realising this trait’s benefits, including disease resistance attributes. However, the existence of unified downstream signaling that favours chlorophyll pigments may operate in the background. Linking this to the alternative pathways may further enhance understanding.

References

Cha K-W, Lee Y-J, Koh H-J et al. (2002) Isolation, characterization, and mapping of the stay green mutant in rice. Theor Appl Genet 104, 526–532.

Cheng J, Kyzy MA, Heide A. et al. (2024) Senescence-Associated Sugar Transporter1 affects developmental master regulators and controls senescence in Arabidopsis. Plant Physiol https://doi.org/10.1093/plphys/kiae430

Duvick D N. (1984) Genetic contribution to yield gains of US hybrid maize 1930-1980. In Genetic contributions to yield gains of five major crop plants (CSSA special pp. 15-47. Ed. W. R. Fehr. Madison: Crop Science Society of America.

Fang C, Li C, Li W. et al. (2014) Concerted evolution of D1 and D2 to regulate chlorophyll degradation in soybean. Plant J 77, 700–712.

Fang Z, Bouwkamp JC and Solomos T. (1998) Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L. Exptl Bot 49, 503–510.

Fu D, Zhou H, Grimm B, et al. (2025) The BCM1-EGY1 module balances chlorophyll biosynthesis and breakdown to confer chlorophyll homeostasis in land plants. Mol. Plant doi: https://doi.org/10.1016/j.molp.2024.11.016.

Gupta R, Anand G, Lorena Pizarro L, et al. (2021) Cytokinin inhibits fungal development and virulence by targeting the cytoskeleton and cellular trafficking. mBio 12:e03068-20.

Jiao B, Meng Q and Lv W. (2020) Roles of stay‑green (SGR) homologs during chlorophyll degradation in green plants. Bot Stud 61, 25.

Qiu T, Wei S, Fang K, …,  Zhao W. et al. (2024) The atypical Dof transcriptional factor OsDes1 contributes to stay-green, grain yield, and disease resistance in rice. Sci Adv 10, eadp0345.

Quirino BF, Reiter W-D, Amasino RD. (2001) One of two tandem Arabidopsis genes homologous to monosaccharide transporters is senescence-associated. Plant Mol Biol. 46:447–457.

Sato, Y. Morita, R. Nishimura, M. et al. (2007) Mendel’s green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway. Proc Natl Acad Sci USA 104, 14169–14174.

Shimoda Y, Ito H and Tanaka A. (2016) Arabidopsis STAY-GREEN, Mendel’s Green Cotyledon gene, encodes magnesium-dechelatase. Plant Cell  28, 2147–2160.

Shim Y, Kang K, An G. et al. (2019) Rice DNA-Binding One Zinc Finger 24 (OsDOF24) Delays leaf senescence in a jasmonate-mediated pathway. Plant Cell Physiol 60, 2065–2076.

Shin D, Lee S, Kim T-H. (2020) Natural variations at the Stay-Green gene promoter control lifespan and yield in rice cultivars. Nat Commun 11, 2819.

Thomas H and Smart CM. (1993) Crops that stay green. Ann Appl Biol 123, 193-219.

Wang Y, Tan J, Wu  Z et al. (2019) STAYGREEN, STAY HEALTHY: a loss-of-susceptibility mutation in the STAYGREEN gene provides durable, broad-spectrum disease resistances for over 50 years of US cucumber production. New Phytol 221:415–430.

Xie W, Xue X, Wang Y, et al. (2024) Natural mutation in Stay-Green (OsSGR) confers enhanced resistance to rice sheath blight through elevating cytokinin content. Plant Biotechnol J  https://doi.org/10.1111/pbi.14540

Zhang G-Q. (2020) Prospects of utilization of inter-subspecific heterosis between indica and japonica rice. Journal of Integr Agric 19, 1–10.