FLOURY ENDOSPERM20 encoding SHMT4 is required for rice endosperm development

To investigate the cytological basis of disrupted storage substance accumulation, we performed scanning electron microscopy (SEM) and light microscopy of endosperm. flo20-1 endosperm was filled with loosely arranged and abnormal starch granules (SGs), compared with tightly packed and sharp-edged SGs in WT (Figure 1c). SHMT4 is required for rice endosperm development. (a) Full view and transverse sections of WT and flo20-1 seeds. (b–d) 1000-brown kernel weight and physicochemical characteristics (b), SEM (c) and light microscopy (d) of WT and flo20-1. (e) Gene structure of SHMT4 and mutation sites of flo20 alleles. Putative splicing sites are underlined. (f) Complementation tests of flo20-1 grains. (g) Expression patterns of SHMT4. (h) Subcellular localization of SHMT4-GFP. (i) Total SHMT activities in WT, flo20-1 and CRI-SHMT3 mutants. (j) The activity analysis of SHMT4 in vitro. (k–l) Y2H (k) and BiFC assay in tobacco (l) showing the interactions of SHMT4 and its homologues. (m) Summary of proteins co-precipitated with SHMT4-GFP, as identified by mass spectrometry. (n) The SHMT activity assay in SHMT4-GFP and GFP transgenic rice. (o) Enzymatic activity of SHMT3 & 5 in vitro. (p) SHMT activity of SHMT4-GST precipitate prepared from different background rice. (q) Verification of SHMT4 interacting with SAMS2 using BiFC and Co-IP assays. (r) Measurement of SAM concentration in WT and flo20-1. (s) Global distribution of DNA methylation levels over promoter, exon, intron and UTR of genes as well as repeat region. (t) RT-qPCR analysis of RPBF, RISBZ3, ONAC025, RISBZ1, ONAC020 and ONAC026 in WT and flo20-1 endosperm at 10 DAF.