The hypothesis that reducing chlorophyll content (Chl) can increase canopy photosynthesis in soybeans was tested using an advanced model of canopy photosynthesis. The relationship among leaf Chl, leaf optical properties, and photosynthetic biochemical capacity was measured in 67 soybean (Glycine max) accessions showing large variation in leaf Chl. These relationships were integrated into a biophysical model of canopy-scale photosynthesis to simulate the intercanopy light environment and carbon assimilation capacity of canopies with wild type, a Chl-deficient mutant (Y11y11), and 67 other mutants spanning the extremes of Chl to quantify the impact of variation in leaf-level Chl on canopy-scale photosynthetic assimilation and identify possible opportunities for improving canopy photosynthesis through Chl reduction. These simulations demonstrate that canopy photosynthesis should not increase with Chl reduction due to increases in leaf reflectance and nonoptimal distribution of canopy nitrogen. However, similar rates of canopy photosynthesis can be maintained with a 9% savings in leaf nitrogen resulting from decreased Chl. Additionally, analysis of these simulations indicate that the inability of Chl reductions to increase photosynthesis arises primarily from the connection between Chl and leaf reflectance and secondarily from the mismatch between the vertical distribution of leaf nitrogen and the light absorption profile. These simulations suggest that future work should explore the possibility of using reduced Chl to improve canopy performance by adapting the distribution of the "saved" nitrogen within the canopy to take greater advantage of the more deeply penetrating light.

The soybean () seed coat has distinctive, genetically programmed patterns of pigmentation, and the recessive mutation can epistatically overcome the dominant and alleles, which inhibit seed color by producing small interfering RNAs (siRNAs) targeting () mRNAs. Small RNA sequencing of dissected regions of immature seed coats demonstrated that siRNA levels cause the patterns produced by the and alleles of the locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. To identify the locus, we compared RNA-seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by siRNAs but different genotypes (homozygous versus homozygous). By examining differentially expressed genes, mapping information, and genome resequencing, we identified a 129-bp deletion in Glyma.11G190900 encoding Argonaute5 (AGO5), a member of the Argonaute family. Amplicon sequencing of several independent saddle pattern mutants from different genetic backgrounds revealed independent lesions affecting , thus establishing Glyma.11G190900 as the locus. Nonfunctional AGO5 from alleles leads to altered distributions of siRNAs, thus explaining how the mutation reverses the phenotype of the seed coat regions from yellow to pigmented, even in the presence of the normally dominant or alleles.

The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat.

The structure of chalcone synthase ( CHS ) gene repeats in different alleles of the I (inhibitor) locus in soybean spawns endogenous RNA interference (RNAi) that leads to phenotypic change in seed coat color of this major agronomic crop. Here, we examined CHS gene copy number by digital PCR and single nucleotide polymorphisms (SNPs) through whole genome resequencing of 15 cultivars that varied in alleles of the I locus ( I , i i , i k , and i ) that control the pattern distribution of pigments in the seed coats. Lines homozygous for the i i allele had the highest copy number followed by the I and i k cultivars which were more related to each other than to the lines with i i alleles. Some of the recessive i alleles were spontaneous mutations, and each revealed a loss of copy number by digital PCR relative to the parent varieties. Amplicon sequencing and whole genome resequencing determined that the breakpoints of several i i to i mutations resulted from nonallelic homologous recombination (NAHR) events between CHS genes located in segmental duplications leading to large 138‐kilobase deletions that erase the structure generating the CHS siRNAs along with eight other non‐ CHS genes. Functional hybrid CHS genes (designated CHS5:1 ) were formed in the process and represent rare examples of NAHR in higher plants that have been captured by examining spontaneous mutational events in isogenic mutant lines.

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