Soybean reduced internode 1 determines internode length and improves grain yield at dense planting

Soybean yields lag behind cereals like rice and wheat, which benefitted from semi-dwarf, lodging-resistant architectures enabling dense planting. In soybean, plant height is determined by node number and internode length; reducing height often lowers node number and pod count, penalizing yield. A key breeding objective is to shorten internodes while maintaining node number to enable high-density planting without yield loss. Gibberellin (GA) pathways and light signaling (CRY1-STF/HY5) regulate soybean internode elongation in response to planting density and blue light levels, but the role of SUPPRESSOR OF PHYA 105 (SPA) proteins in soybean internode control was unknown. This study asks whether a SPA homolog regulates internode length and whether such alleles can improve yield under dense planting. The authors identify and characterize a short-internode mutant (rin1), determine its genetic basis, elucidate its molecular mechanism via interactions with HY5 homologs (STF1/2) and GA metabolism, and test its agronomic utility under high-density field conditions.

The work builds on Green Revolution genes in cereals: rice sd1 (GA20-oxidase 2) and wheat Rht-B1b/Rht-D1b (DELLA variants) that reduce plant height and enable dense planting with higher yields and nitrogen-use efficiency. In soybean, GA pathway genes influence height, and a CRY1–STF(HY5)–gibberellin module adjusts internode elongation to light quality under high density by modulating GA2ox expression and GA1 levels. In Arabidopsis, COP1 forms a complex with SPA proteins to ubiquitinate and degrade HY5, repressing photomorphogenesis; SPA1 can also independently reduce HY5 in the light. SPA proteins have conserved WD-repeat and coiled-coil domains necessary for function. However, whether SPA proteins impact plant height and internode length in soybean had not been explored. This study situates RIN1 (a SPA3a homolog) within these pathways, linking SPA–HY5-like (STF1/2) interactions to GA metabolism and internode control.

- Mutant identification: Generated a γ-ray–irradiated mutant library in elite cultivar Heinong 35 (HN35); identified a compact, short-internode mutant named rin1 with increased per-plant yield. - Genetic mapping: Crossed rin1 with elite cultivar Heihe 43 (HH43) to produce an F2 (HNH) population. Performed GBS-based QTL mapping for plant height and internode length, locating rin1 on chromosome 12. F2:3 validation confirmed the locus. - Fine mapping and cloning: Constructed residual heterozygous lines segregating only at rin1; delimited the locus to a 76.31 kb region with seven annotated genes. Identified Glyma.12G224600.1 (SPA3a) as the only gene with coding variation between parents. Detected two nonsynonymous SNPs (CDS positions 223 and 2654) distinguishing cultivars and a premature stop at position 2521 in rin1 causing truncation. Named the wild-type alleles RIN1Wm82 and RIN1HH43. - Functional validation: Created CRISPR/Cas9 knockouts rin1CR1 and rin1CR2 in Wm82; confirmed absence of off-target edits in other SPA homologs. Phenotyped under long-day conditions; knockout and rin1 mutants displayed dwarfism with fewer nodes and shorter internodes. Developed near-isogenic lines (NIL-RIN1HH43 and NIL-rin1) for field validation. - Expression analyses: Assessed RIN1 expression by RT-qPCR and in situ hybridization in shoot apical meristems (SAM) across vegetative stages; performed diurnal expression in NILs. - Protein interaction and stability assays: Tested physical interactions between RIN1 alleles and STF1/2 by yeast two-hybrid, in vitro pull-down, and BiFC in tobacco. Measured STF1/2 protein abundance and degradation using in vivo (western blot across light/dark; MG132 treatments) and cell-free degradation assays with extracts from NILs. - Genetic interaction: Generated stf1 stf2 double mutants (CRISPR) and STF2 overexpression lines; constructed NIL combinations to evaluate epistasis and genetic dependence of RIN1 on STF1/2. - Transcriptional regulation: Quantified GA2ox7a and GA2ox7b expression in SAM of NILs, Wm82, rin1, and rin1CR; used in situ hybridization. Conducted dual-luciferase reporter assays in Nicotiana to test effects of RIN1 alleles on GA2ox promoters. - Hormone measurements and treatments: Quantified GA profiles (GA1, GA8) in SAM by LC-MS/MS. Applied exogenous GA3 at V1 stage to NILs and measured internode lengths dose-responsively. - Haplotype analysis: Analyzed natural variation in RIN1 using resequencing of 1,295 accessions; associated haplotypes with phenotypes across three field locations. - Field trials: Evaluated agronomic traits and yield of HN35 and rin1 across planting densities (250k, 350k, 450k plants/ha) in Harbin; measured plant height, node number, internode length, grain yield per plant, and plot yield.

- Gene identification: rin1 maps to a 76.31 kb interval on chromosome 12; the causative gene is Glyma.12G224600.1 (SPA3a). The rin1 allele carries a premature stop at CDS position 2521 truncating the protein; additional nonsynonymous variants exist at positions 223 and 2654 among cultivars. - Function and phenotype: CRISPR knockouts (rin1CR1/CR2) phenocopy rin1 with pronounced dwarfism, fewer nodes, and shorter internodes under long-day conditions. NIL-rin1 lines confirm reduced plant height and internode length in the field. - Expression pattern: RIN1 is highly expressed in the shoot apical meristem, leaf primordia, and axillary meristems, peaking at 10 days after emergence, consistent with a role in early internode and node development. - Protein interactions and stability: RIN1 physically interacts with STF1 and STF2 (soybean HY5 homologs) in yeast, in vitro, and in planta. RIN1 promotes STF1/2 degradation via the 26S proteasome in both light and dark; STF1/2 are stabilized in NIL-rin1 extracts. The rin1 protein still binds STF1/2 but with weaker interaction. - Transcriptional regulation of GA metabolism: RIN1 suppresses GA2ox7a and GA2ox7b expression in SAM. In NIL-rin1 and rin1CR, GA2ox7a/b are up-regulated; dual-luciferase assays show functional RIN1 represses GA2ox promoters, while rin1CR loses repression and rin1 exhibits reduced function. STF1/2 promote GA2ox7a/b expression; RIN1 effects are partially dependent on STF1/2 genetic background. - Hormone levels and rescue: NIL-rin1 has reduced bioactive GA1 and increased GA8 in SAM. Exogenous GA3 application rescues internode length differences between NILs, supporting GA-mediated control. - Genetic dependence: The rin1 effect on internode length is attenuated in the stf1 stf2 double mutant background, indicating partial dependence of RIN1 on STF1/2. - Agronomic performance: Across planting densities (250k–450k plants/ha), rin1 plants are shorter with shorter internodes yet maintain higher grain yield per plant compared to HN35. Plot-level yield of rin1 exceeds HN35, with the largest advantage at the highest density (450k plants/ha), demonstrating suitability for dense planting. - Natural variation: Three RIN1 haplotypes (H1–H3) differ in flowering time but not internode length; knockout alleles reduce height and promote flowering, suggesting potential to tailor maturity and architecture.

The study establishes RIN1 (a SPA3a homolog) as a positive regulator of node number and internode elongation in soybean and reveals a mechanistic pathway wherein RIN1 interacts with HY5-like factors (STF1/2) to promote their proteasomal degradation, thereby repressing GA2ox7a/b expression, maintaining higher bioactive GA levels, and promoting internode elongation. The rin1 allele weakens this regulation, increasing STF abundance, elevating GA2ox expression, lowering GA1, and shortening internodes. Crucially, shortening internodes without substantial loss of nodes enables compact architecture advantageous for high-density planting. Field trials show rin1 increases plot yield at higher densities, addressing the breeding objective of boosting soybean population yield without sacrificing reproductive sites. The genetic interactions demonstrate RIN1 functions upstream of STF1/2 and partially depend on them, integrating light signaling with GA metabolism to fine-tune stem architecture. These findings bridge photomorphogenic E3 ligase components (SPA-like) with agronomic traits in soybean and provide a deployable allele for modern dense-planting systems and intercropping.

This work identifies SPA3a as RIN1, defines its role in controlling soybean internode length via STF1/2-mediated regulation of GA2ox genes and GA homeostasis, and demonstrates that the rin1 allele confers compact architecture and higher yield under dense planting. The study delivers both mechanistic insight (RIN1–STF–GA2ox module) and a practical breeding resource (rin1) for improving soybean population yield and potential soybean–maize intercropping. Future research should evaluate the effects of rin1 and complete knockout alleles on nodulation and nitrogen fixation under low-nitrogen conditions, test whether knockout alleles can combine early maturity with dense-planting yield gains, and explore environment-by-genotype interactions across broader latitudes and management regimes.

- The impact of rin1 and knockout alleles on nodulation and biological nitrogen fixation, particularly under low-nitrogen conditions, remains untested and could influence yield outcomes. - Flowering-time effects vary by genetic background; mechanisms controlling flowering vs. internode length may be independent and require further dissection. - Field validations were conducted at specific sites and seasons; broader multi-environment trials are needed to generalize yield advantages. - Quantitative yield advantages are presented at plot scale without detailed multi-year, multi-location statistical analyses for all agronomic traits.