Additional file 2: of Characterization of S40-like proteins and their roles in response to environmental cues and leaf senescence in rice

Table S1. Cis elements in the promoters of S40 genes in rice, HvS40 and AtS40–3. Promoter regions of 840 bp upstream of HvS40 and 1000 bp upstream of Ats40–3, and rice S40 genes were analyzed with the use of the PLACE program. W-box: Binding site for WRKY TFs; ERE: Elicitor response element; MYB: Myeloblastosis; LREs: Light regulated elements; MYC: Myelocytomatosis; ABRE: Abscisic acid responsive elements; Dof: DNA-binding with one finger; PRE: Pathogen response elements; SURE: Sulfur response elements; DRE/CRT: Dehydration response elements/C-repeat; LTR: Low temperature response; ARF: Auxin response factor; DPBFCOREDCDC3: BZIP TFs binding core sequence; G-box plus G: TF OsIRO2-binding core sequence. Table S2. Characteristics of rice S40 proteins. Characteristics of rice S40 proteins including theoretical isoionic point (PI), molecular weight (MW), Number of amino acids, instability index, aliphatic index and GRAVY (Grand Average of Hydropathy) predicted by ProtParam tool ( http://web.expasy.org/protparam/ ). Figure S1. Exon-intron structures of S40 genes in rice genome. Yellow color shows CDS (exon), Blue color shows UTR (untranslated regions) while normal line represents introns. Figure S2. Distribution of OsS40 genes on rice chromosomes. Chromosome Map Tool was used to located genes on chromosome. Figure S3. Amino acid sequences of the four Arabidopsis, two rice and one barley protein of group I compared to the sequence of the barley HvS40 protein. The conserved DUF584 domain sequence was highlighted in black and 100% identical residues in grey. Figure S4. Conserved motifs in HvS40, AtS40–3 and OsS40 proteins. a Motif structures for the proteins were determined using MEME search tool. Grey lines represent the non-conserved sequence. Each motif is indicated by a coloered box numbered at the bottom. b Moti logo obtained by MEME program. The overall height of each stack represents the degree of conservation at each position, while the height of letters within each stack indicates the relative frequency of amino acids. The motifs, numbered 1–10, were displayed in different colored boxes. Figure S5. Semi qRT-PCR expression analysis of the sixteen OsS40 genes at different growth stages of flag leaves, labeled as 90DAG, 97DAG, 104DAG, 111DAG) and 118DAG. DAG (Days After Germination). Figure S6. Semi qRT-PCR expression analysis of the sixteen OsS40 genes at different nitrogen concentrations. Genes marked with * indicate the differentially expressed genes that were further analyzed by quantitative real-time PCR. Figure S7. Semi qRT-PCR expression analysis of the sixteen OsS40 genes during dark induced leaf senescence. Detached leaves from 4-week-old rice seedlings were incubated in the deionized water in darkness for 2 days (D). As a control, the detached leaves were incubated with water at the same time in a light/dark regime (L). Genes marked with * indicate the four differentially expressed genes that were further analyzed by Real-Time PCR. Figure S8. Detached leaves of four weeks old rice plants were treated with different concentrations (50uM, 100uM, 200uM) of ABA, SA, MeJa and IAA. Treatment with water was used as a control. The effect of the treatment was shown by the yellowing of leaves, which initiated after 48 h of treatment with 200 μM concentration of these hormones. Figure S9. Semi qRT-PCR expression analysis of the sixteen OsS40 genes in respond to ABA, SA, MeJA or IAA treatment. Treatment with water was used as a control. Among them, eight genes showed altered expression at different time of treatment. Genes marked with * indicate the eight differentially expressed genes that were further analyzed by quantitative real-time PCR. Figure S10. Semi qRT-PCR expression analysis of the sixteen OsS40 genes in responds to M. oryzae infection. As a control the rice seedlings were sprayed with the 0.02% (w/v) Tween 20 solution only (Mock). After inoculation, the leaves were collected at 24 hpi, 48 hpi, 72 hpi, 96 hpi and 108hpi for RNA extraction. Genes marked with * indicate the ten differentially expressed genes that were further analyzed by quantitative real-time PCR. hpi, hours post-inoculation. Figure S11. Immunoblot analysis of C-terminal GFP-tagged OsS40 memebers transiently expressed in rice protoplasts. The corresponding GFP-tagged OsS40 proteins with expected molecular sizes are pointed out with an arrow. The actin protein and Ponceau S staining were used to check the loading level. (ZIP 14091 kb)