2.4. Gene expression level analysis of Solanum lycopersicum SerR and AspRs To compare the gene expression levels, we analyzed publicly available RNA-Seq data of Solanum lycopersicum obtained from GenBank SRA database (Accession No. SRP068096). The numbers of RNA-Seq reads for Solanum lycopersicum SerR, AspR1, and AspR2 were counted at seven mature organs (flesh, peel, seed, leaf, petals, buds and root) and calculated the RPKM values (Supplemental Table S2). The Solanum lycopersicum SerR and AspR1 genes are constantly expressed throughout all mature organs suggesting that these proteins may have an essential function involved in D- amino acid metabolism. The expression level of the SerR is about twice that of the AspR1 except in seed, which may be related to the fact that the enzyme activity of the AspR is more than 1000 times higher than that of the SerR (Table 2 and Supplemental Table S2). In contrast to the SerR and AspR1 genes, the RNA-Seq reads corresponding to the AspR2 gene are hardly detected in any of the mature organs, suggesting the AspR2 gene may be expressed under limiting conditions or may have become a pseudogene in the recent past (Supplemental Table S2). In order to further examine the gene expression of SerR and AspRs in different plant organs and tissues, we use the TomExpress (http://tomexpress.toulouse.inra.fr/ aboutTomExpress; Zouine et al., 2017) and the tomato functional genomic database (TFGD; http://ted.bti.cornell.edu/cgi-bin/TFGD/ digital/home.cgi; Fei et al., 2011). These web-based transcriptomic platforms are using the publicly available RNA-Seq data sets and provide the gene expression profile discriminated by Solyc ID which is based on the gene locus in Solanum lycopersicum genome. The Solyc ID of Solyc08g008010 precisely corresponded to the SerR gene, while Solyc08g008000 corresponded to both adjacent genes AspR1 and AspR2. Therefore, it is not possible to distinguish the gene expression level of AspR1 and AspR 2 in TomExpress and TFGD, so we referred to the expression level of Solyc08g008000 as that of AspRs. However, gene expression level of the Solyc08g008000 is expected to be mainly due to the AspR1 gene, on the basis of above-mentioned data analysis using RNA-Seq data of seven mature organs (Supplemental Table S2). The gene expression pattern of Solanum lycopersicum SerR and AspRs from RNA-Seq data (Supplemental Table S2) and results from TomExperss and TFGD do not always agree with each other. For example, TomExpress (Supplemental Table S3) and TFGD (Supplemental Table S4) show that AspR expression is predominantly in seeds but AspR expression seems to be quite uniform among different tissues (Supplemental Table S2). The AspRs expression in seeds tends to decrease as the fruit matures (Supplemental Tables S3 and S4). However, the seeds used for RNA-Seq analysis in the Supplemental Table S2 was derived from red ripe fruit. We thought that the expression level of the AspRs in seeds is low in the Supplemental Table S2. There are some other differences in gene expression level, because different development stage samples were used in each database. Thus, we only describe the gene expression patterns supported by a large number of datasets. A comparison of gene expression level of Solanum lycopersicum SerR and AspRs using 95 RNA-Seq data points from TomExpress (Supplemental Tables S3) and 90 RNA-Seq data points from TFGD (Supplemental Table S4) revealed the following findings (Fig. 3). Solanum lycopersicum SerR is expressed in all investigated samples, and its expression level is significantly higher in the shoot apical meristem and leaf primordium, which are both active cell division tissues (Fig. 3; Supplemental Table S3). The ubiquitous expression of the SerR gene and its increased expression level in active cell division tissues has also been reported in Arabidopsis thaliana (Fujitani et al., 2006; Sugimoto et al., 2009). This expression pattern suggests that plant SerR has multiple roles, and it might be related to the fact that SerR is a bifunctional enzyme, producing both D- serine and pyruvate. The expression of Solanum lycopersicum AspRs is detected in all tissues, similar to SerR, but AspR expression level is much lower than that of SerR in all tissues except seed, ovule, and the surrounding ovule tissues (Fig. 3; Supplemental Table S4). The expression level of AspRs increased sharply in ovule after anthesis and became 20 times higher, especially in embryo, compared to that of SerR, suggesting that D- aspartate is synthesized by AspR and it may be involved in plant embryo development.

Published as part of Uda, Kouji, Edashige, Yumika, Nishimura, Rie, Shikano, Yuuna, Matsui, Tohru, Radkov, Atanas D. & Moe, Luke A., 2020, Distribution and evolution of the serine / aspartate racemase family in plants, pp. 1-8 in Phytochemistry (112164) (112164) 169 on pages 4-5, DOI: 10.1016/j.phytochem.2019.112164, http://zenodo.org/record/8293818