Additional file 2: Table S3. of Chloroplast proteome response to drought stress and recovery in tomato (Solanum lycopersicum L.)

Identification details for the proteins observed as differentially represented by 2D-DIGE. Reported are spot number, protein accession/description, MASCOT score, total and unique peptides detected, identification rank and unicity, experimental and theoretical peptide mass value, peptide charge state, peptide mass error, number of peptide missed cleavage(s), peptide score, peptide expectation value and sequence, including amino acid(s) modification. (XLSX 1109 kb

Additional file 1: Table S1. of Chloroplast proteome response to drought stress and recovery in tomato (Solanum lycopersicum L.)

Nucleotide sequence of primers used for qRT-PCR analyses. Figure S1. Representative 2D-DIGE showing the resolution of about 2600 protein spots from tomato chloroplasts in IEF using pH 3-10 NL 18 cm strips, followed by 12% T SDS-PAGE. Spot visualization was obtained with a Typhoon fluorescence scanner. Differentially represented spots further subjected to nLC-ESI-LIT-MS/MS analysis are highlighted. A Cy-2 labeled pooled sample mixture was used as an internal standard for quantitative measurements. Table S2. Listed are data referring to spot number, NCBI protein accession number, protein description, MASCOT score value, theoretical and experimental molecular mass and pI values, total and unique peptides detected, sequence coverage (%), EMPAI score value, fold change in drought stressed plants with respect to control, and fold change in recovered drought stressed plants with respect to well-watered control of the same age. In addition, data referring to protein spots not showing significant quantitative variations are reported in italics and are highlighted in green. Protein species showing either an incoherent quantitative trend or a constant trend among the experimental conditions are highlighted in blue. (PDF 2377 kb

Expression patterns of <i>TOPP1</i> and <i>AtI-2</i> as indicated by <i>promoter</i>::<i>GUS</i> activities.

<p>(A) to (H) GUS staining of transgenic plants expressing <i>ProTOPP1</i>::<i>GUS</i> and <i>ProAtI-2</i>:<i>GUS</i> at different growth stages. Typical staining images of germinating (A) and 4-day-old (B) seedlings, aerial tissues (C) and roots from 2-week-old seedlings (D), rosette leaves (E) and cauline leaves (F) and flowers (G) as well as siliques (H) from 6-week-old plants are shown. Scale bar = 1 mm.</p

RNA-seq analysis shows the overlapping pattern of transcriptome changes between <i>topp1</i> and <i>ati-2</i> in response to ABA.

<p>(A) The Venn diagram represents the number of genes that were significantly changed at least 1.2-fold in <i>topp1</i> or <i>ati-2</i> mutant plants than wild-type after ABA treatment (FDR value < 0.05; fold change ≥1.2). Differentially expressed ABA responsive genes were identified by DESeq (P-value < 0.05; fold change > 4 after ABA treatment). (B) Heatmap depiction of the ABA-responsive gene expressions in wild type, <i>topp1</i> and <i>ati-2</i>. Heatmap was created by heatmap.2 function of gplots package in R. (C) Gene ontology enrichment analysis of the differentially expressed (DE) genes co-regulated by <i>TOPP1</i> and <i>AtI-2</i>. The statistically enriched GO categories were shown based on the number and percentage of DE genes (p value < 0.05).</p

TOPP1 and AtI-2 physically interact with SnRK2s.

<p>(A) TOPP1 interacts with SnRK2s in Y2H assay. (B) AtI-2 interacts with SnRK2s in Y2H assay. TOPP1 or AtI-2 was fused to the GAL4-activating domain (AD) and SnRK2s were fused to the GAL4-DNA binding domain (BD). Interaction in Y2H assay was determined by yeast growth on media lacking Leu, Trp and His in the absence or presence of ABA. Dilutions (10⁻¹, 10⁻² and 10⁻³) of saturated cultures were spotted onto the plates and photographs were taken after 3 days. (C) TOPP1 interacts with SnRK2s in split-luciferase complementation (split-LUC) assay. (D) AtI-2 interacts with SnRK2s in split-LUC assay. (E) TOPP1 interacts with SnRK2.6 in Co-IP assay. (F) AtI-2 interacts with SnRK2.6 in Co-IP assay. (G) Alt-2 enhances the interaction between TOPP1 and SnRK2.6 in split-LUC assay. The split-LUC assays were repeated at least three times. Anti-LUC western blot shows the levels of the indicated fusion proteins.</p

Effect of TOPP1 and AtI-2 on ABA and salt sensitivity.

<p>(A) ABA sensitivity of <i>topp1</i> and <i>ati-2</i> mutants as well as the <i>TOPP1</i> and <i>AtI-2</i> over-expression lines during germination and early seedling growth. (B) Quantification of the green cotyledon expansion rates of indicated genotypes in response to ABA and NaCl. The green cotyledon expansion rate of indicated genotypes was recorded at 7 days after germination. At least 50 seeds per genotype were sterilized and germinated on ½ MS agar plates with or without 0.5 μM ABA or 100 mM NaCl in each replicate. Seedlings with green cotyledon expansion were counted. Data indicate mean values± SD from three replicates. Significance between the mean values were analyzed with Student’s <i>t</i> test (* P˂ 0.05). (C) The ABA-mediated inhibition of seedling growth of wild type, and <i>TOPP1</i> and <i>AtI-2</i> over-expression lines. Photographs were taken at 7 days after seedlings transfer to ½ MS plates, 10 μM ABA or 20 μM ABA containing MS plates. At least ten 3-day-old seedlings from indicated genotypes were transferred and root length was recorded after 7 days. The experiments were repeated at least three times with similar results. (D) Quantification of the fold changes of primary root length of wild type, <i>topp1</i>, <i>ati-2</i>, and <i>TOPP1</i> and <i>AtI-2</i> over-expression lines at 7 days after transfer. Values are means ± SD (<i>n</i> = 30). Significance between the mean values were analyzed with Student’s <i>t</i> test (* P˂ 0.05, ** P˂ 0.01).</p

A proposed model of TOPP1- and AtI-2-mediated ABA signaling pathway.

<p>TOPP1-AtI-2 complex can modulate ABA signaling through physical interactions with PYL11 and SnRK2s. Binding of TOPP1 and AtI-2 with SnRK2.6 can suppress the kinase activity of SnRK2.6. Such suppression can be released at least partially by PYL11 in the presence of ABA.</p

TOPP1 and AtI-2 physically interact with PYLs.

<p>TOPP1 interacts with PYLs in Y2H assay. AtI-2 interacts with PYLs in Y2H assay. TOPP1 or AtI-2 was fused to the GAL4-activating domain (AD) and PYLs were fused to the GAL4-DNA binding domain (BD). (A) TOPP1 interacts with PYL11 in split-LUC assay. (B) AtI-2 interacts with PYL11 in split-LUC assay. Alt-2 enhances the interaction between TOPP1 and PYL11 in split-LUC assay. Anti-LUC western blot shows the levels of the indicated fusion proteins. (C) The split-LUC assay were repeated at least three times with consistent results.</p

TOPP1 and AtI-2 suppress SnRK2.6 signaling.

<p>(A) TOPP1 and AtI-2 suppressed the activity of SnRK2.6, which can be released by PYL11 in the presence of ABA. ABA signaling pathway was reconstituted by co-expression of TOPP1, AtI-2, PYL11, SnRK2.6 and ABF2 in wild type (Col-0) protoplasts. The induction of <i>RD29B</i>::<i>LUC</i> was used as the ABA-responsive reporter, while <i>ZmUBQ</i>::<i>GUS</i> was used as the control for transformation efficiency. After co-transformation, protoplasts were incubated for 4 h under light without (close bars) or with 5 μM ABA (open bars). Relative luciferase activities were statistically summarized from at least three biological replicates. Error bars indicate SD. (<i>n</i> = 3). (B) Increased SnRK2.2/3/6 activities in <i>topp1</i> and <i>ati-2</i> mutants in in-gel kinase assay. The total proteins were extracted from 4-day-old seedlings without or with ABA treatment. Histone was used as a substrate for SnRK2.2/3/6. Coomassie blue staining indicates the equal loading. The experiment was repeated at least three times with similar results.</p

TOPP1 and AtI-2 inactivate SnRK2.6 <i>in vitro</i>.

<p>(A) TOPP1 diminishes SnRK2.6-catalyzed ABF2 phosphorylation. (B) AtI-2 does not affect SnRK2.6 activity. Recombinant protein MBP-SnRK2.6 and GST-ABF2 were incubated with GST-AtI-2. The phosphorylation of ABF2 was not reduced in the presence of AtI-2. (C) AtI-2 enhances the inactivation of SnRK2.6 by TOPP1. MBP-SnRK2.6 and GST-ABF2 were incubated with GST-TOPP1, or GST-AtI-2 or both GST-TOPP1 and GST-AtI-2. The SnRK2.6 activity was indicated by the radio-activities of GST-ABF2. (D) PYL11 partially prevents the inhibition of SnRK2.6 by TOPP1 or TOPP1/AtI-2. MBP-SnRK2.6 and GST-ABF2 were incubated with GST-TOPP1, or the combination of GST-TOPP1 and GST-AtI-2. GST-PYL11 was introduced with or without ABA. At least three independent experiments were performed in (A) to (D) with similar results.</p