PH13 improves soybean shade traits and enhances yield for high-density planting at high latitudes

Soybean is a globally important oilseed and protein crop with demand projected to double by 2050. Traditional soybean cultivars are short-day plants with strong photoperiod sensitivity, limiting their adaptation across latitudes. While long-juvenile traits have enabled expansion to lower latitudes, high-latitude regions (over 40°N) offer substantial untapped production potential but pose challenges due to long-day photoperiods that extend maturity and induce exaggerated stem elongation (ESE), causing lodging and yield loss, especially under high-density planting or intercropping. Several flowering-time loci (E1–E4, Tof5+, Tof11, Tof12) have facilitated northern adaptation by modifying maturity, yet the genetic basis controlling plant height—critical for adaptation to high latitudes and dense planting—remains poorly understood. Light conditions, especially low blue light under canopy shade or high density, trigger ESE; GmCRY1s repress LBL-induced ESE by stabilizing bZIP transcription factors STF1/2. This study aims to identify plant height loci that mitigate ESE and elucidate mechanisms enabling shade resistance and high-density planting at high latitudes.

Prior work has identified flowering-time genes (E1–E4, Tof5+, and PRR homologs Tof11/Tof12) instrumental for adapting soybean to northern latitudes by adjusting maturity. However, fewer plant height loci have been dissected, despite numerous QTLs reported near PH13 on chromosome 13. Shade avoidance in soybean is mediated by light signaling components: blue light receptors GmCRY1s stabilize bZIP transcription factors STF1/2 to repress LBL-induced ESE, and GmCOP1 orthologs influence plant height via degradation of such factors. In Arabidopsis, SPA proteins partner with COP1 E3 ubiquitin ligases to regulate photomorphogenesis by targeting transcription factors for degradation. This body of literature suggests conserved COP1/SPA mechanisms may regulate shade responses and height, motivating discovery of soybean SPA homologs modulating STF1/2 abundance.

- Germplasm and phenotyping: 2214 genotyped soybean accessions were evaluated for plant height across ten locations over two or three years. Best linear unbiased predictions (BLUPs) were computed using a mixed linear model accounting for line, location, year, and interactions. - GWAS/TWAS: GWAS used 540 improved cultivars genotyped at 3,469,934 SNPs (MAF >5%) with FarmCPU, controlling population structure via top three SNP principal components; Bonferroni threshold α=0.05 (p<1.44E-08). TWAS used RNA-seq from 488 accessions (tissues above cotyledonary node at V2) with FarmCPU and a mixed linear model in GAPIT for confirmation; expression PCs controlled structure; expressed features had TPM>0.1; Bonferroni correction applied. - Haplotype and structural variant analysis: PH13 coding sequences were examined in 1254 accessions via resequencing and PCR. A 5404 bp Ty1/Copia-like retrotransposon insertion at the start of exon 5 was identified; exonic SNPs (e.g., Gm13-37816013) defined three major haplotypes (H1–H3). Immunoblotting assessed protein truncation. - Functional validation: CRISPR/Cas9 targeted PH13 in Williams 82 (W82H1) and Tianlong 1 (TL1H3) backgrounds using multiple gRNAs; independent mutants were obtained. Overexpression lines (35S::PH13-3xFlag) were generated in TL1H3. Near-isogenic lines (NILH1/NILH3) were created by crossing W82H1 and TL1H3 and selecting segregants. - Expression and subcellular localization: RT-qPCR with exon 3 and 3’UTR primers examined PH13 transcript regions; diurnal expression patterns of PH13 and GmCOP1a/b were assessed. YFP fusions tested subcellular localization of PH13 haplotypes in Arabidopsis protoplasts. - Protein interaction assays: Yeast two-hybrid mapped interactions between PH13 haplotypes/domains and GmCOP1a/b; β-galactosidase assays quantified interaction strength. Co-immunoprecipitation in Nicotiana benthamiana tested PH13–GmCOP1b interactions in vivo. - Paralog analysis: The PH13 paralog Glyma.12G224600 (PHP) was identified; CRISPR generated php single mutants and ph13/php double mutants (phd) in TL1H3. STF1/2 protein abundance was measured across genotypes and diurnally. - Shade and light treatments: Seedlings of WT and mutants were grown under white light (WL), low blue light (LBL), or WL plus far-red (low R:FR) for 15 days after de-etiolation under long-day conditions. PAR ~500 μmol m−2 s−1. - Field trials and agronomic assessments: Multi-location field trials in Beijing (39°54′N), Changchun (43°53′N), and Harbin (45°70′N). Planting density trials in Changchun at 30/20/10/5 cm spacing (~66,700/100,000/200,000/400,000 plants ha−1) with three replicates. Relay intercropping trials with maize in Harbin. Traits measured at R8 included plant height, internode length, branches, node number, center of gravity height, pods per plant, lodging rate, yield per plant, and yield per plot. Statistical tests included unpaired two-tailed Student’s t-tests and two-way ANOVA with Tukey tests as appropriate.

- Discovery of PH13: GWAS identified 11 loci and TWAS identified 7 genes for plant height; PH13 (Glyma.13G276700) on chromosome 13 was the only gene supported by both analyses and lies within a repeatedly reported height QTL region. The leading SNP (Gm13-37757704) was in high LD (R^2=0.86) with an exonic SNP in PH13. - Natural haplotypes and structural variant: Three major PH13 haplotypes (H1–H3) were defined; H3 contains a 5404 bp Ty1/Copia-like retrotransposon inserted at exon 5, truncating the C-terminal WD40 domain. Immunoblotting confirmed a ~10 kDa smaller PH13H3 protein. Accessions with PH13H3 had significantly reduced plant height compared to H1/H2 across ten locations over multiple years (e.g., p=1.83×10−21 between H1 and H3). - Expression and mechanism: The insertion abolishes transcription of the carboxyl terminus but not upstream regions. PH13 and GmCOP1a/b show diurnal expression peaking at dawn. PH13H1/H2 strongly interact with GmCOP1a/b, whereas PH13H3 interacts weakly; the WD40 domain enhances interaction strength. Reduced interaction with GmCOP1s in H3 leads to increased STF1/2 protein accumulation. - STF1/2 accumulation: In NILH3 versus NILH1 under long days, STF1/2 protein levels increased by 39–144% over a diurnal cycle, peaking mid-day; in the ph13/php double mutant (phd-1), STF1/2 abundance increased at least 3-fold, especially at night. - Functional validation of PH13: CRISPR loss-of-function ph13 mutants in W82 (H1) showed >30% reduction in plant height due to decreased internode length and fewer nodes; overexpression of PH13 increased plant height. In TL1H3, ph13 mutants had ~10% reduction, indicating residual function of PH13H3. NILH3 plants were shorter than NILH1. - Selection for high latitudes: PH13H3 is absent in wild G. soja, rare in landraces (1.4%), but frequent in improved cultivars (32.7%). PH13H3 accessions are enriched at higher latitudes; in China, the proportion of PH13H3 among cultivars rose to 48.7% above 40°N (76 of 78 PH13H3 cultivars located above 40°N). - Role of paralog PHP and double mutants: PH13 and its paralog PHP (Glyma.12G224600; 96.6% aa similarity) act cooperatively. Phenotypes progressed TL1H3 > ph13 > php > phd in reduced height. The phd double mutant exhibited decreased internode length without reduced node number and thicker stems under all light regimes. - Shade resistance and yield under density: ph13/php knockout abolished LBL- and low R:FR-induced ESE; phd mutants were shade-resistant and lodging-resistant under all tested planting densities. In density trials (up to 400,000 plants ha−1), phd height was insensitive to increasing density, showed no lodging, and exceeded TL1H3 in per-plant yield; plot yields of phd increased significantly with density while TL1H3 was unchanged or decreased. Compared to elite cultivar Jiyu202 at 400,000 plants ha−1, phd had thicker stems, lower lodging, more branches and pods, and 15.8% higher grain yield per plant. - Intercropping performance: In maize–soybean relay intercropping at Harbin, phd mutants were shorter, had no lodging (vs nearly 100% for TL1 and 40–80% for other elites), and achieved plot yields comparable to elite cultivars LK317 and LK18-842. - Proposed model: The Ty1/Copia insertion in PH13 reduces PH13–GmCOP1 interaction, elevates STF1/2 abundance, and reduces internode elongation. Gene editing of PH13 and PHP produces an ideal shade-resistant, lodging-resistant architecture suitable for high-density planting at high latitudes.

This study elucidates a genetic basis for plant height adaptation to high-latitude environments and dense planting in soybean. PH13, a SPA-like WD40 protein, modulates stem elongation via interaction with GmCOP1 E3 ligases to regulate STF1/2 abundance. A naturally occurring Ty1/Copia insertion truncates PH13 (H3 haplotype), weakening PH13–GmCOP1 interactions and elevating STF1/2, thereby reducing plant height. The H3 allele has been strongly selected in improved cultivars at higher latitudes, indicating its utility in breeding for reduced lodging and improved adaptation. However, PH13H3 alone may be insufficient to prevent ESE under extreme long-day conditions; combining PH13H3 with early-flowering loci likely optimizes adaptation. Gene editing of PH13 and its paralog PHP produced double mutants with ideal shade-resistant architecture—shorter plants with thicker stems and maintained node number—conferring insensitivity to density-induced shade cues, reduced lodging, and increased yield under high-density and intercropping systems. Mechanistically, the WD40 domain enhances the PH13–GmCOP1 interaction, and diurnal expression patterns suggest coordination with photoperiod to modulate STF1/2 turnover. Differences from Arabidopsis (e.g., PH13 subcellular distribution and WD40 contribution to COP1 interaction) highlight species-specific nuances in COP1/SPA signaling controlling architecture.

The work identifies PH13 as a major determinant of soybean plant height and shade response, uncovers a natural Ty1/Copia insertion (H3) that truncates PH13 and reduces COP1 interaction to elevate STF1/2, and shows that this allele has been selected for high-latitude adaptation. Functional genomics revealed cooperative roles of PH13 and its paralog PHP; simultaneous knockout generates a shade-resistant, lodging-resistant architecture enabling high-density planting and intercropping with improved yield. These findings provide actionable alleles and a gene-editing strategy for breeding high-yielding soybean cultivars suited to high latitudes and dense planting. Future work could integrate PH13/PHP alleles with early-flowering loci for higher-latitude environments and evaluate performance across broader germplasm and environments.

- The PH13H3 allele alone may be insufficient to fully mitigate ESE at high latitudes, as evidenced by TL1H3 exhibiting severe ESE and lodging; combining with early-flowering loci is suggested. - Functional validations and field trials were conducted in specific genetic backgrounds (W82 and TL1) and environments in China; broader testing may be needed to generalize across diverse germplasm and agroecologies. - The mechanistic studies focus on interactions with GmCOP1s and STF1/2; additional downstream targets or pathways influencing plant architecture under varying light qualities may exist and were not exhaustively explored.