Natural variations at the Stay-Green gene promoter control lifespan and yield in rice cultivars

Global food demand is rising with population growth, but rice yield gains have stagnated in recent years. Asian rice predominantly comprises two subspecies, indica and japonica, which differ markedly in lifespan and senescence; indica exhibits earlier senescence and a rapid life cycle. While early senescence may be adaptive under r-selection in tropical, multi-cropping systems, it can be detrimental to grain filling and yield in temperate single-cropping regions. Senescence and stay-green traits influence crop productivity, and loss-of-function stay-green (sgr/ossgr) mutants delay chlorophyll degradation but often do not maintain photosynthetic function or yield advantages. The study aims to identify the genetic basis underlying the subspecies-specific senescence and lifespan differences, focusing on the Stay-Green gene (OsSGR), and to test whether natural allelic variation—particularly in promoter regions—explains these phenotypes and can be leveraged to improve yield.

Previous work shows senescence is a regulated process affecting nutrient remobilization, grain filling, and yield. Stay-green traits in crops like maize and sorghum correlate with yield improvements, but in rice, ossgr mutants are nonfunctional stay-green: chlorophyll persists without maintaining photosynthetic capacity or yield benefits. OsSGR encodes Mg2+-dechelatase, a key enzyme in chlorophyll catabolism. Genetic studies (QTLs and GWAS) have identified loci and natural variants impacting chlorophyll content and stay-green traits in rice. Arabidopsis PRR9 and rice OsNAP are known regulators of age-dependent senescence. Together, these studies motivated examining OsSGR allelic variation—especially promoter polymorphisms—as a potential driver of subspecies-specific senescence dynamics and agronomic performance.

The authors conducted QTL mapping using an F2:3 population (141 lines) from a cross between indica IR72 and japonica Junam (JN), quantifying leaf senescence via colorimetric assays (ACA) and chlorophyll meters during grain filling. GBS-based linkage analysis identified significant QTLs (LOD threshold 4.3). Fine mapping of the strongest locus on chromosome 9 used 6,349 BC5F2 lines derived from backcrossing to JN, delimiting a 26-kb region between markers C9-10 and C9-12 containing OsSGR (LOC_Os09g36200), Harpin-induced protein 1, and OsPRR95. Functional validation included: (1) activation-tagging lines for OsSGR and OsPRR95 in japonica Dongjin; (2) CRISPR/Cas9 OsSGR knockouts in indica Kasalath and RNAi lines; and (3) OsSGR overexpression lines (indica and japonica coding sequences under maize Ubi promoter). To test whether coding variation affects function, in vitro Mg2+-dechelatase assays (wheat-germ expression) and in planta assays (tobacco infiltration) compared enzymatic activities of indica, japonica, and ossgr proteins. Promoter contribution was examined by measuring OsSGR transcript induction during senescence and via transient luciferase reporter assays in rice protoplasts comparing 2-kb promoters from IR72 (indica) and JN (japonica). Association analyses used 105 O. sativa accessions across five subgroups; 2-kb OsSGR promoter regions were sequenced to define polymorphisms and haplotypes, and chlorophyll levels at 6 weeks after heading were measured; site-wise associations were evaluated with t-tests and empirical multiple-testing correction. Phylogenetic analyses of promoter haplotypes included O. nivara and O. rufipogon and an expanded WGS dataset (n=1477) to assess relationships and selective sweep signals (πω/πρ in 20-kb windows). Breeding tests introgressed japonica OsSGR promoter alleles (haplotype 8) into elite indica cultivars (IR72, Milyang21, Milyang23) to create near-isogenic lines (NILs) through repeated backcrossing; a reciprocal JN-NIL carrying the indica promoter (haplotype 1) was also generated. Field trials in Korea assessed senescence progression (ACA, chlorophyll), transcript levels (qRT-PCR of OsSGR and OsNAP), photosynthetic performance (Fv/Fm, net CO2 assimilation LI-6850), relative growth rate (dry mass log-growth between timepoints), grain-filling rate, and grain yield per plant.

• QTL mapping identified multiple loci controlling flag and second-leaf senescence; the strongest locus on chromosome 9 (shared by both leaves) was fine-mapped to a 26-kb interval containing OsSGR. • Functional tests: OsSGR activation lines showed accelerated senescence and reduced yield; OsSGR knockouts in indica delayed chlorophyll loss in leaves and panicles. OsPRR95 activation had no effect on senescence. • Coding-region SNPs between indica and japonica OsSGR alleles did not account for phenotypic differences: overexpressing either allele caused similar early senescence; in vitro and in planta assays showed comparable Mg2+-dechelatase activity for indica and japonica proteins, while ossgr lacked activity. • Promoter effects: OsSGR expression rose earlier and to higher levels in indica than japonica during senescence; in protoplasts, the IR72 (indica) promoter drove 3.1-fold higher reporter activity than the JN (japonica) promoter. • Across 105 rice accessions, sequencing the 2-kb OsSGR promoter revealed 21 polymorphisms defining eight haplotypes; chlorophyll content differed by haplotype, with indica haplotype 1 having substantially lower chlorophyll than japonica haplotype 8. Sixteen of 21 sites significantly associated with chlorophyll, 15 coinciding with indica–japonica differences. • Phylogeny grouped the indica promoter with O. nivara and the japonica promoter with O. rufipogon, consistent with subspecies ancestry; no evidence for a domestication selective sweep around OsSGR. • A distinct Dof transcription factor–binding motif (AAAAGCTC insertion) is present in the indica promoter and associates with low chlorophyll, suggesting a cis-regulatory mechanism for earlier/higher OsSGR induction. • Agronomic impact: Introgressing the japonica OsSGR promoter into elite indica cultivars delayed senescence, sustained photosynthetic competence (higher chlorophyll, improved net CO2 assimilation), and increased yield. Yield per plant increased to 39.6 g in IR72-NIL (from 35.9; +10.6%), 32.7 g in Milyang21-NIL (from 29.0; +12.7%), and 32.6 g in Milyang23-NIL (from 29.1; +12.0%). Grain-filling rates increased accordingly, and IR72-NIL maintained higher RGR especially 5–7 weeks after flowering. Conversely, replacing japonica JN’s promoter with the indica promoter (JN-NIL) caused earlier senescence, reduced photosynthesis, and decreased yield to 27.6 g per plant (−10.1%) with a 6.5% reduction in grain-filling rate.

The study demonstrates that natural promoter variation at OsSGR underlies key differences in senescence timing and lifespan between indica and japonica rice. Earlier and stronger OsSGR induction in indica accelerates chlorophyll degradation and senescence, aligning with rapid-cycling strategies favored in multi-cropping environments. In contrast, slower OsSGR induction in japonica extends functional photosynthesis during grain filling, benefiting yield in single-cropping systems. The absence of functional differences in OsSGR proteins across subspecies and the strong association of promoter polymorphisms (including a potential Dof-binding motif insertion) with chlorophyll content implicate cis-regulatory evolution as the driver of phenotypic divergence. Population analyses tracing promoter haplotypes to O. nivara (indica) and O. rufipogon (japonica) support an evolutionary origin predating domestication, without evidence of a selective sweep. Importantly, swapping promoter alleles altered senescence kinetics and photosynthetic performance, translating into predictable yield gains or losses. These findings provide a mechanistic and evolutionary framework for manipulating senescence timing via promoter alleles to optimize grain filling and productivity according to cropping system and environment.

Promoter polymorphisms at the non-regulatory enzyme gene OsSGR causally control senescence timing, lifespan, and yield outcomes in rice subspecies. By fine-mapping, functional assays, and association/phylogenetic analyses, the study shows that indica-type promoters drive earlier/higher OsSGR expression and faster senescence, while japonica-type promoters delay senescence and extend photosynthetic competence. Introgressing the japonica OsSGR promoter into elite indica cultivars consistently increased grain yield by about 10–13% under Korean field conditions. These naturally occurring promoter alleles provide an immediately deployable breeding strategy to fine-tune senescence and enhance productivity. Future work should pinpoint the causal promoter polymorphism(s), validate the role of the identified Dof-binding motif and its cognate transcription factors, and evaluate performance across diverse environments and cropping systems.

The precise causal promoter variant(s) among the 16 associated polymorphisms were not functionally dissected, and the proposed involvement of a Dof-binding motif remains correlative without direct TF binding/functional validation. Yield benefits were demonstrated in specific genetic backgrounds and environments (Korean fields), so broader environmental and genotype-by-environment validation is needed. While OsSGR knockouts delayed chlorophyll loss, they did not improve yield, underscoring the complexity of functional versus nonfunctional stay-green phenotypes; the study did not identify regulatory networks beyond OsSGR that might modulate senescence. No selective sweep signal was detected, but this does not exclude more subtle selection on regulatory variants.