*2.1. yl Plants Show Yellow Leaves and Abnormal Photosynthesis at the Seedling Stage*

The *yl* mutant was identified from the soybean cultivar Jindou 23 mutagenized with ethyl methane sulfonate (EMS). The *yl* mutant plants exhibited conspicuous yellow leaves (Figure 1A). Chlorophyll (Chl) analysis revealed that the Chl *a*, *b* and total Chl contents of wild type (Jindou 23) leaves were 1.15 mg/g, 0.89 mg/g and 2.04 mg/g, respectively (Figure 1B). In contrast, the Chl *a*, *b* and total Chl contents of *yl* leaves were 0.86 mg/g, 0.34 mg/g and 1.20 mg/g, respectively (Figure 1B). In addition, the Chl *a*/*b* ratio of *yl* leaves was significantly increased compared to that of the wild type (Figure 1C). To evaluate whether the photosynthesis of *yl* leaves was affected, we measured the maximum photochemical efficiency of photosystem II (PSII), defined as the Fv/Fm and net photosynthesis rate. The *yl* leaves displayed obviously decreased Fv/Fm values (wild type, 0.73; *yl*, 0.62) (Figure 1D). Moreover, the net photosynthesis rate of *yl* leaves was 9.03 μmol CO2 m−2s−1, which was only 60% that of wild type leaves (14.93 μmol CO2 m<sup>−</sup>2s−1) (Figure 1E). Therefore, the mutation in *yl* causes not only a reduction in chlorophyll contents but also abnormalities in photosynthesis.

**Figure 1.** Phenotypic characterization of the wild type and *yl* mutant plants. (**A**) Two-week-old wild type (left) and *yl* (right) seedlings. (**B**) Chlorophyll contents of the third leaf of the wild type (left) and *yl* (right) plants. (**C**) The Chl *a*/*b* ratio of the wild type (left) and *yl* (right) leaves. (**D**) The maximum photochemical efficiency of PSII measured by the Fv/Fm chlorophyll fluorescence ratio in the wild type

(left) and *yl* (right) leaves. (**E**) The net photosynthesis rate of the wild type (left) and *yl* (right) plants. Error bars (**B**–**E**) represent the mean ± SD (*n* ≥ 3); \*\* indicates a significant difference at the 0.01 level. The *p* values were calculated by Student's t-test. Bar = 5 cm in (**A**).

We further performed transmission electron microscopy (TEM) to compare the chloroplast ultrastructure between wild type and *yl* leaves. No differences in the number of chloroplasts per cell or chloroplast size were detected between the wild type and *yl*. However, the number of grana (stacked thylakoids) were slightly reduced in the *yl* chloroplasts (Figure 2A–D), indicating that *yl* chloroplasts were probably less functional than wild type chloroplasts.

**Figure 2.** Chloroplast ultrastructure in the wild type and *yl* leaves. (**A**,**B**) Transmission electron micrographs (TEM) of chloroplasts in the wild type (**A**) and *yl* (**B**) leaves. (**C**,**D**) Magnified images of a chloroplast in the wild type (**C**) and *yl* (**D**) leaves. Arrows show grana. Bar = 0.5 μm in (**A**,**B**) and 200 nm in (**C**,**D**).
