Supplementary Data Captions
Supplementary Table S1. Primers used for RT PCR
Supplementary Table S2. Primers used for real time PCR
Supplementary Figure S1 Sequence analysis of the SbSAP14 and phylogenomic relationship between SbSAP14 and O. sativa/S. bicolor SAP proteins. a The amino acids at N-terminal A20 and the C-terminal AN1 type zinc-finger domains are conserved in the SbSAP14 protein. b Amino acid sequence alignment of the AN1/A20 domain with other SAP proteins. Conserved residues are shaded. Gaps indicated by dashes have been introduced to maximize alignment. Number shown on the right side indicates a position of amino acid residues. c Phylogeny of SbSAP14 and related proteins. The tree was derived from the AN1/A20 domain peptide sequence. Branch length numbers are shown.
Supplementary Figure S2 Localization of 35S::SbSAP14-GFP fusion protein in root cells of transgenic Arabidopsis. a The root tissues of Arabidopsis seedlings (5 to 7-day-old) were treated without (unplasmolyzed) or with 0.5 M mannitol (plasmolyzed) for at least 30 min, and the reported fluorescence emitted from the fusion protein was observed under a confocal microscope. The cells within the dashed red box in a was magnified and shown in b. White arrows indicated that 35S::SbSAP14-GFP fusion protein formed round spots with almost identical size throughout the cytoplasm compartment. GFP green fluorescence, DIC (differential interference contrast) and merged images were shown. Scale bars = 10 µm.
Supplementary Figure S3 Construct of transgenic rice lines overexpressing the SbSAP14 gene. The binary vector pCAMBIA1390 was used to construct transgenic rice lines overexpressing the SbSAP14 gene that was under the control of maize Ubi promoter (a). The SbSAP14 transcript levels were analyzed by RT-PCR in WT and in the heterozygous T1 generation transgenic lines (b) and in the homozygous T2 transgenic lines (c) that were used for the following tolerant test. Rice actin amplification was used as an internal control. WT and transgenic plants (2-week-old) were treated without (d) or with (e) 250 mM NaCl for 3 d at 28oC, then the typical phenotypic pictures were photographed. Scale bar = 5 cm.
Supplementary Figure S4 At 3 d after treated without (CK) or with 250 mM NaCl at 28oC, chlorophyll a content was tested between SbSAP14 transgenic lines and WT seedlings as described in Materials and Methods. Each value is the mean ± SD of three replicates. FW is for fresh weight.
Supplementary Figure S5 Proline content and expression of proline synthesis-related genes in WT and transgenic plants under salt stress. a WT and transgenic seedlings of two-week-old were treated without (Control) or with 200 mM NaCl at 28oC for 3 d and proline content was determined as described in Materials and Methods. Each value is the mean ± SD of three replicates. b WT and transgenic seedlings of two-week-old grown at 28oC were treated without (Control) or with 200 mM NaCl for 24 h and total RNA was isolated and subjected to semi-quantitative RT-PCR analysis of proline synthesis-related genes as described in Materials and Methods. Similar results were observed in three independent experiments.
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