*2.4. Expression Pattern and Protein Subcellular Localization of YL*

We performed quantitative real-time RT-PCR (qRT-PCR) to examine the expression of *YL* among various tissues. *YL* was expressed in all tissues tested, with the highest expression in trifoliate leaves, intermediate expression in meristem, nodules, expanded leaves, cotyledons, and young pods and weak expression in roots, stems, flowers, and young seeds (Figure 5A). The expression pattern of *YL* was consistent with the function of *YL* in affecting leaf color.

Using the Predotar and TargetP programs, the YL protein was predicted to localize in chloroplasts [32,33]. To obtain experimental verification of this localization, we expressed a YL-GFP fusion protein under the 35S promoter in *Nicotiana benthamiana* leaves. The green fluorescence signals of the YL-GFP fusion protein were observed in the chloroplasts of epidermal cells (Figure 5B,C).

**Figure 4.** The YL protein contains an RNA recognition motif. (**A**) Amino acid sequence of YL. Position of the *yl* (asterisk) mutation is exhibited. The RNA recognition motif is underlined. (**B**) Partial alignment of the amino acid sequences of YL and its homologous proteins. The aligned sequences are from *Glycine max* (YL, Glyma.20G187000), *Arabidopsis thaliana* (AtORRM1, At3G20930), *Zea mays* (ZmORRM1, GRMZM5G899787), *Oryza sativa* (Os02g49820), *Medicago truncatula* (Medtr1g092860) and *Phaseolus vulgaris* (Phvul.007G103400). The red rectangle represents the conserved alanine residue in the RNA recognition motif.

**Figure 5.** Expression pattern and subcellular localization of *YL*. (**A**) Relative expression levels of the *YL* gene in various soybean tissues. The real-time RT-PCR assays were performed in three biological

replicates. qRT-PCR values for *YL* are normalized with the actin and ATP synthase genes. Error bars represent the SD. (**B**,**C**) Subcellular localization of the YL-GFP fusion protein (**B**) and GFP (**C**) in epidermal cells of *Nicotiana benthamiana* leaves. Bar = 20 μm in (**B**) and 50 μm in (**C**).
