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Impact of Reduced Chlorophyll Levels in Leaves on Soybean Yield, Seed Composition, Pod/Seed Photosynthesis, and Chlorophyll Levels in Pod and Seed Tissues

Sarah I. JonesiD, Samantha S. StutziD, Eray Atalay, Yu Wang, Donald R. OrtiD, Young B Cho

Abstract

Soybean, a widely cultivated leguminous crop valued for its protein, amino acids, and oil, faces the challenge of maintaining protein levels, which have an inverse correlation with yield. Reducing leaf chlorophyll levels could increase seed protein levels without compromising yield; however, this is yet to be tested. Therefore, to understand the impacts of low chlorophyll mutations on soybean yield and seed composition, we screened and compared 25 low chlorophyll soybean mutants to their 11 dark green parents. PI548210 (Lincoln mutant) demonstrates a higher concentration of protein without affecting yield compared to its dark green parent PI548362 (Lincoln), suggesting it as a good candidate for further large-scale field trials. PI547555 (Y11/y11, Clark mutant) demonstrates a lower concentration of oil without impacting yield, alongside lower gross photosynthesis, but with chlorophyll levels in the pod and seed tissues that are comparable to its dark green parent PI548533 (Clark). These findings are consistent with the oil concentration of the soybean being influenced by pod and seed photosynthesis, which is correlated with pod height and row spacing. Chlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed. SIGNIFICANCEO_LIPI548210 (Lincoln mutant), one of twenty-five low chlorophyll soybean mutants, demonstrates a higher concentration of soybean protein without affecting yield compared to its dark green parent (Figure 1 and Table 1). C_LIO_LIPI547555 (Y11/y11, Clark mutant), a low chlorophyll soybean mutant, demonstrates a reduced concentration of soybean oil without impacting yield, alongside lower gross photosynthesis in pod and seed tissues compared to its dark green parent (Figures 3 and Table 2). These findings suggest that the oil concentration of the soybean is influenced by pod and seed photosynthesis, which is in turn influenced by pod height and row spacing (Figure 2). C_LIO_LIChlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed (Figure 5-6). C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/744892v1_fig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1364f0forg.highwire.dtl.DTLVardef@80657borg.highwire.dtl.DTLVardef@186af1forg.highwire.dtl.DTLVardef@1532c64_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Two low chlorophyll mutants are as healthy as their dark green parents. Lincoln and its low chlorophyll mutant, left; Clark and its low chlorophyll mutant, known as Y11/y11, right. It can be seen by eye that the plants have low chlorophyll (light green/yellow leaves) but a similar growth habit to their dark green parents. See Supplemental Figures 1-4 for contrast, where low chlorophyll mutants are stunted in growth compared to their dark green parents. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@1877bc4org.highwire.dtl.DTLVardef@1ce85bforg.highwire.dtl.DTLVardef@1aecde2org.highwire.dtl.DTLVardef@14ee288org.highwire.dtl.DTLVardef@1fa5cb9_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONComparison of seed yield, weight, seed composition between low chlorophyll mutants and their dark green parents. ANOVA is used with linear mixed model (random effect = block, fixed effect = variety). Least squares mean is used to compare. For yield and seed composition, N=4 blocks. For leaf chlorophyll (SPAD), N=40. Yield is average yield per plant (g). n.s. = not significant. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/744892v1_fig3.gif" ALT="Figure 3"> View larger version (26K): org.highwire.dtl.DTLVardef@1dda2ceorg.highwire.dtl.DTLVardef@1ebbed6org.highwire.dtl.DTLVardef@3de91aorg.highwire.dtl.DTLVardef@92c4c1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3.C_FLOATNO Light response curve of low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Rates of net and gross photosynthesis of low chlorophyll (white) and dark green parents (black) pods under field conditions. Each dot represents a value (n=4) {+/-}SE. We assumed that the seeds greatly inhibited the transmittance of light through the pod and used photosynthetic photon flux density for a single-side. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@1b3822dorg.highwire.dtl.DTLVardef@2774ecorg.highwire.dtl.DTLVardef@6d6866org.highwire.dtl.DTLVardef@e123a2org.highwire.dtl.DTLVardef@ea977f_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONPod photosynthetic parameters for low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Photosynthesis was measured 1 September through 15 September 2021 at the University of Illinois Energy Farm in Urbana, IL, USA. The statistical analysis was done using ANOVA with linear mixed model (alpha=0.05). N=4 {+/-} SEM for Clark and N=3 {+/-} SEM for Y11. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/744892v1_fig2.gif" ALT="Figure 2"> View larger version (23K): org.highwire.dtl.DTLVardef@1804595org.highwire.dtl.DTLVardef@584540org.highwire.dtl.DTLVardef@12aa13eorg.highwire.dtl.DTLVardef@1c0049c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533) differ in concentration of seed oil, which interacts with height of pod and row spacing. The box plots show the median (central line), the lower and upper quartiles (box) and the minimum and maximum values (whiskers). The statistical analysis was done using ANOVA with linear mixed model (n=3 blocks, alpha=0.05). Least squares mean is used to compare. N.s., non- significant in the analysis. A. Concentration of oil in low chlorophyll mutant seeds from the upper canopy decreased by 4% compared to the dark green parent (18.2% vs 19%) while there was no difference between them in the seeds from the lower canopy (20.2% vs 20.6%). B. Schematic layout of 2013 field setting showing two different row spacings. C. Concentration of oil in low chlorophyll mutant decreased by 2% in 38cm spacing (21.4% vs 22%) while there was no difference in 19cm spacing (21.3% vs 21.7%) in 2013 field. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/744892v1_fig5.gif" ALT="Figure 5"> View larger version (22K): org.highwire.dtl.DTLVardef@16ec397org.highwire.dtl.DTLVardef@eb1e6dorg.highwire.dtl.DTLVardef@1a8fcforg.highwire.dtl.DTLVardef@1d76437_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5C_FLOATNO (greenhouse). Correlation between the level of leaf chlorophyll (x-axis: SPAD reading) and the level of immature pod or seed chlorophyll (y-axis, mg/g DW). Line represents the linear regression model. R-squared is a coefficient of determination, the percentage of the response variable variation that is explained by the linear model. Pod is labeled by the fresh weight of seeds it contained. A. Level of chlorophyll of 25-100mg pod (n=18). B. Level of chlorophyll of 100-200mg pod (n=17) . C. Level of chlorophyll of 25-100mg seed (n=17). D. Level of chlorophyll of 100-200mg seed (n=20). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/744892v1_fig6.gif" ALT="Figure 6"> View larger version (28K): org.highwire.dtl.DTLVardef@1f482e8org.highwire.dtl.DTLVardef@1be65f0org.highwire.dtl.DTLVardef@1169fa4org.highwire.dtl.DTLVardef@4b092b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Levels of gene expression in chlorophyll synthesis pathway. A. CHL common pathway genes; Glutamyl-tRNA reductase (GluTR). Glutamate 1- semialdehyde aminotransferase (GSA-AT). ALA dehydratase (ALAD). Uroporphyrinogen III synthase (UROS). Uroporphyrinogen III decarboxylase (UROD). Protoporphyrinogen IX oxidase (PPO). B. Mg branch; Mg-chelatase (Mgch). Magnesium-protoporphyrin IX monomethyl ester cyclase (MPEC). Protochlorophyllide reductase (POR). 3,8-divinyl protochlorophyllide a 8-vinyl-reductase (4VCR). Heme pathway; Ferrochelatase (FECH). Heme oxygenase (HO). Phytochromobilin synthase (HY). Data come from Severin et al (2010). RPKM, reads per kilobase per million mapped reads. DAF, days after flowering. The source seed is experimental line A81-356022 which was generated by introgressing G. soja (PI468916) into G. max (A81-356022). C_FIG

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (19 Aug 2026) · CC BY 4.0

Among 25 low-chlorophyll soybean mutants, a Lincoln mutant raised seed protein without yield loss, while Clark-derived Y11/y11 lowered seed oil alongside reduced pod gross photosynthesis.

Raising soybean seed protein is hard because protein trades off against oil and yield. Chlorophyll holds roughly 14% of leaf nitrogen, so cutting it might free nitrogen for seed filling; earlier work showed a low-chlorophyll soybean keeps canopy photosynthesis, but its effect on seed composition, and the role of pod/seed photosynthesis in seed oil, were untested or unclear.

Key results

  1. SuggestedAcross the screen, leaf chlorophyll did not predict yield or seed composition. Comparing each mutant with its own parent turned up one Lincoln mutant (PI548210) with more seed protein and less oil than its dark green parent (PI548362) at comparable yield.

    In the 25 mutants, 2021 leaf SPAD spanned 11 to 51, and leaf chlorophyll barely tracked yield (R^2 0.07), oil (0.1) or protein (0.04); 9 of 25 mutants matched parental yield. PI548210 vs PI548362 (N=4 blocks): leaf chlorophyll 51% lower (p = 0.001), oil 16.7% lower (p < 0.001), protein 5.4% higher (p < 0.009); cysteine and methionine also significantly higher. · Two low chlorophyll mutants exhibit comparable…; DISCUSSION; Tables

  2. ShownThe Clark Y11/y11 mutant (PI547555) had slightly less seed oil than Clark (PI548533), with similar yield and protein. The gap appeared in upper-canopy seed and at wide row spacing, and was not significant in lower-canopy seed or in dense 19 cm rows.

    2021: leaf chlorophyll 62% lower (p <0.001), oil 3% lower (p = 0.03). Upper canopy oil 18.2% vs 19%; lower canopy 20.2% vs 20.6% (n.s.). 2013 seed: 38 cm rows 21.4% vs 22% (2% lower, p = 0.006); 19 cm rows 21.3% vs 21.7% (n.s.); n=3 blocks. · Two low chlorophyll mutants exhibit comparable…; The impact of low leaf chlorophyll on soybean…; Figure legends

  3. ShownClark pods had almost twice the gross photosynthesis of Y11/y11 pods, but net photosynthesis did not differ. Pod/seed chlorophyll content, photosystem II (PSII) operating efficiency and simulated under-canopy light did not explain the shortfall.

    Gross photosynthesis, Clark vs Y11/y11: 6.1 vs 3.1 μmol CO2 m^-2 s^-1 at 2000 μmol m^-2 s^-1 and 4.5 vs 2.6 at 300 μmol m^-2 s^-1 (N=4 vs N=3 pods). Dark respiration was nearly twice as high in Clark. Pod Fq'/Fm' was 0.384 in the mutant vs 0.317 in Clark; seed Fq'/Fm' and pod/seed chlorophyll did not differ significantly. · The Clark low chlorophyll mutant (Y11/y11) has a…; Pod and seed photosynthesis measurements; Sample collection and chlorophyll fluorescence…; Tables

  4. ShownLeaf chlorophyll poorly predicted chlorophyll in immature pods and seeds across varieties. Pods and seeds contained far less chlorophyll than leaves in every genotype.

    Leaf SPAD vs extracted pod/seed chlorophyll across up to 20 varieties at two seed stages: R^2 0.088 to 0.28 (greenhouse panels n=17 to 20); field data also showed no strong correlation. Pod and seed chlorophyll was 5- to more than 100-fold lower than in leaves. · The level of leaf chlorophyll does not correlate…; Levels of chlorophyll and expression of genes in…; Chlorophyll measurement; Figure legends

  5. SuggestedIn public RNA-seq data, most genes of the shared chlorophyll pathway and the Mg branch are expressed far less in pods and seeds than in young leaves, while heme-branch genes follow a different pattern. This may explain why leaf and reproductive-tissue chlorophyll are decoupled in the mutants.

    58 genes encoding 16 enzymes. Expression in pods/seeds was up to 30-, 21-, 11-, 53- and 46-fold lower than in young leaves for GluTR, GSA-AT, PPO, Mg-chelatase and POR. ChlI1a (Glyma.13G305600, mutated in Y11/y11): 106 RPKM in young leaves, 33 to 41 in immature pods, 2 to 8 in immature seeds. · Levels of chlorophyll and expression of genes in…; DISCUSSION; Transcriptome data analysis; Figure legends

Methods, data and reproducibility

Field screen (2021, plus archived 2013 row-spacing seed) comparing low-chlorophyll mutants with dark green parents for yield and near-infrared (NIR) seed composition using mixed models. Follow-up work used attached-pod gas-exchange light curves, PSII fluorescence imaging, tissue chlorophyll extraction and reanalysis of public soybean RNA-seq for chlorophyll-pathway genes.

  • Low-chlorophyll mutants (mostly natural, from the USDA Soybean Germplasm Collection) and dark green parents, 2021 Urbana IL field, 4 blocks of about 10 plants each · Glycine max (soybean) · n = 36 varieties (25 mutants, 11 parents)
  • Lincoln low-chlorophyll mutant and its dark green Lincoln parent (the focal protein comparison) · Glycine max (soybean) · n = 4 blocks for yield/composition; SPAD N=40 · PI548210; PI548362
  • Heterozygous Y11/y11 Clark mutant (Mg-chelatase ChlI1a) and its dark green Clark parent, 2021 field · Glycine max (soybean) · n = 4 blocks; canopy-position comparison n=3 blocks · PI547555; PI548533
  • Archived 2013 field seed of Clark and Y11/y11 grown at 19 cm and 38 cm row spacing · Glycine max (soybean) · n = 3 blocks per row spacing per variety · PI548533; PI547555
  • Light-response curves on attached pods (gas exchange), field, September 2021 · Glycine max (soybean) · n = 4 pods per genotype measured; Table 2 reports N=4 Clark, N=3 Y11/y11
  • Chlorophyll fluorescence imaging of immature pod-halves and seeds (seed fresh weight 100-200 mg) · Glycine max (soybean) · n = Clark 26 pod-halves, 35 seeds; Y11/y11 8 pod-halves, 11 seeds
  • Leaf SPAD vs extracted immature pod and seed chlorophyll at two seed stages, greenhouse and field · Glycine max (soybean) · n = up to 20 varieties (16 in field)
  • Public soybean RNA-seq atlas (Severin et al. 2010) from SoyBase: roots, flowers, nodules, young leaves, and seed and pod developmental stages · Glycine max line A81-356022 (G. soja PI468916 introgressed) · n = seven seed stages, three pod stages
Methods & tools
LI-6800 gas exchange system with clear-top chamber (LI-COR), Perten DA7250 NIR spectroscopy (PerkinElmer), SPAD-502 Plus chlorophyll meter (Konica Minolta), CF Imager chlorophyll fluorescence imager (Technologica), Ethanol chlorophyll extraction with microplate spectrometry, ImageJ, R 4.0.5, lme4 1.1-27.1 (lmer), lmerTest 3.1-3 (difflsmeans), SoyBase SoySeq transcriptome resource, Canopy photosynthesis simulation (model not specified)
Data & accessions
https://soybase.org/soyseq/
Reagents & environment
R 4.0.5; lme4 1.1-27.1 (lmer with REML and Satterthwaite degrees of freedom); lmerTest 3.1-3 (difflsmeans); Tukey or Dunnett multiple-comparison adjustment.
Reproducibility gaps
The data statement points to the article and SI Appendix, but the supplemental figures, tables and data file are not in the provided text. No analysis code is released, and the canopy simulation is not described.
Limitations (6)
  • The trials were small-scale. The authors say the Lincoln protein trait still needs testing in larger, multi-location trials, and its causal mutation has not been identified. · stated by the authors
  • The Lincoln mutant was picked up from post-harvest seed composition, so there are no immature-tissue chlorophyll or physiology data for it. The genetic cause of its low-chlorophyll phenotype, as for most mutants screened, is unknown. · stated by the authors
  • Why Y11/y11 pods have lower gross photosynthesis is unresolved. Chlorophyll, PSII efficiency and light were ruled out; lower Vcmax or Jmax in pod/seed tissue is proposed but was not measured. · stated by the authors
  • The authors cannot rule out that more light under the mutant canopy raised temperature, which could itself lower seed oil. · stated by the authors
  • Gross pod photosynthesis was calculated by adding dark respiration to net rates. Net rates did not differ while Clark respiration was nearly twice as high, so the gross difference mostly reflects respiration, and it rests on only N=4 vs N=3 pods. · noted in this summary
  • The tissue-specific expression argument relies on public RNA-seq from a single dark green experimental line (A81-356022), not from the mutants, so applying it to the low-chlorophyll lines is an inference. · noted in this summary

Who should read:Read this if you study soybean protein/oil trade-offs, nitrogen-saving strategies based on reduced chlorophyll, or how pod/seed photosynthesis and canopy position shape seed oil.

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