1. Carbonylation and Loss-of-Function Analyses of SBPase Reveal Its Metabolic Interface Role in Oxidative Stress, Carbon Assimilation, and Multiple Aspects of Growth and Development in Arabidopsis 
Xun-Liang Liu, Hai-Dong Yu, Yuan Guan, Ji-Kai Li, Fang-Qing Guo 
Molecular Plant 
Volume 5, Issue 5, Pages (September 2012)
DOI: /mp/sss012
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Identification of SBPase as a Carbonylated Protein in Detached Leaves Exposed to MV Treatments.
(A, B) Western blot analysis showing carbonyl levels of SBPase protein in wild-type leaves in response to control conditions (A) and MV treatment (10 μM, 2 h) (B) with an anti-DNP antibody. Total protein extracts were prepared from the fully expanded detached leaves of 21-day-old wild-type plants and DNPH-derivatized proteins were analyzed by 2D SDS–PAGE. Protein spots were identified by MALDI–TOF/TOF MS analysis.
(C, D) Representative tandem mass spectra were showed according to precursor ions with m/z values of and , corresponding respectively to (C) peptide MFSPGNLR, spanning residues M260 to R267 of protein SBPase (At3g55800), and (D) peptide NEIIRFEETLYGTSR, spanning residues N369 to R383 of protein SBPase (At3g55800).
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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3. Figure 2
ROS Levels and CO2 Assimilation Rates of Detached Leaves Exposed to MV Treatments.
(A) H2O2 levels were visualized by staining with 3,3'-diaminobenzidine in the fully expanded detached leaves treated with MV (10 μM) for 180 min. Leaves were detached from 21-day-old wild-type plants.
(B) CO2 assimilation rates were measured in the fifth leaves detached from 21-day-old wild-type plants after MV treatments (2 h, 10 μM) (n = 6, mean ± SE). Error bars indicate standard errors (SE).
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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4. Figure 3 Enzymatic Activity of Oxidized SBPase Protein.
(A) Coomassie blue staining of GST (lane1) and GST–SBPase (lane 2) purified from Escherichia coli expressing GST–SBPase fusion protein.
(B) Western blot analysis of the purified GST–SBPase (lane 1) and GST (lane 2) with an anti-SBPase polyclonal antibody.
(C) Characterization of enzymatic activity and substrate specificity of GST–SBPase.
(D) Carbonyl levels of GST–SBPase protein monitored using anti-DNP immunoassay when treated with hydroxyl radical generated via the Fenton reaction. Samples (2 μg each) were separated by SDS–PAGE and analyzed on protein gel blots with an anti-DNP antibody.
(E) SBPase activity of the purified GST–SBPase protein challenged with hydroxyl radical generated via the Fenton reaction (n = 3, mean ± SE).
(F) Effect of adding DTT on SBPase activity of the purified GST–SBPase protein challenged with hydroxyl radical generated via the Fenton reaction (n = 3, mean ± SE). Error bars indicate standard errors (SE).
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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5. Figure 4 Characterization of sbp Mutant.
(A) Schematic diagram of SBPASE gene showing the T-DNA insertion site. Open box indicates 5' or 3' UTR; closed box indicates ORF. Exons (boxes) and introns (lines) were determined by a comparison of the genomic and cDNA sequences. T-DNA is inserted in the transition site of the fifth exon and fifth intron in sbp mutant (SALK_130939).
(B) Phenotypes of wild-type and sbp mutant plants at days 21, 36, and 70 after germination.
(C) RT–PCR analysis of SBPASE mRNA levels in the fully expanded leaves of wild-type and sbp mutant plants.
(D) Western blot analysis of SBPase protein levels in the fully expanded leaves of wild-type and sbp mutant plants with a polyclonal antibody against SBPase.
(E) Enzymatic activities of SBPase in the fully expanded leaves of wild-type and sbp mutant plants (n = 3, mean ± SE).
(F) CO2 assimilation rate of the fully expanded leaves detached from wild-type and sbp mutant plants (n = 6, mean ± SE). Error bars indicate standard errors (SE).
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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6. Figure 5 Complementation of sbp Mutant with SBPASE Genomic DNA.
(A) Phenotypes of sbp mutant plants complemented by a 4.2-kb SBPASE genomic fragment. Plants of wild-type, sbp mutant, and the complemented sbp mutant were photographed at days 30 (upper panel) and 45 (lower panel) after germination.
(B) RT–PCR analysis of SBPASE mRNA levels in the fully expanded leaves of wild-type, sbp mutant, and the complemented sbp mutant plants.
(C) Protein gel blot analysis of SBPase protein levels in the fully expanded leaves of wild-type, sbp mutant, and the complemented sbp mutant plants with a polyclonal antibody against SBPase. Equal protein loading was confirmed with antiserum against α-Tubulin.
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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7. Figure 6
Analysis of pSBPASE:GUS Expression in Transgenic Plants.Transgenic Arabidopsis plants harboring pSBPASE:GUS constructs were analyzed by GUS-staining assay.(A) GUS-staining patterns of a representative 10-day-old transgenic seedling.(B–D) Close-up images of a cotyledon (B), hypocotyl-root junction (C), and primary root tip (D) of the seedling shown in (A).(E–H) GUS-staining patterns in inflorescence (E), silique (F), opening flower (G), and stamens (H).(I-L) GUS-Staining patterns of transgenic plant at days 14(I), 21(J), 35(K) and 45(L) after germination.
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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8. Figure 7
Leaf and Flower Development in Wild-Type and sbp Mutant Plants.
(A) Rosette leaves of wild-type and sbp mutant plants on bolting.
(B) Phenotypes of wild-type and sbp mutant flowers.
(C) Phenotypic comparison of individual sepals, petals, stamens, and gynoecium detached from the representative flowers of wild-type and sbp mutant plants.
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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9. Figure 8
Effects of the SBPASE Mutation on Mesophyll Cell Size and Chloroplast Biogenesis.
(A) Confocal microscopic fluorescence signals (green) of SBPase–GFP in guard cells peeled from the transgenic Arabidopsis plants carrying p35S–SBPASE cDNA–GFP constructs.
(B–D) Analysis of chloroplast numbers in mesophyll cells of wild-type and sbp mutant fully expanded leaves cross-sectioned and examined by TEM (B), by isolating individual mesophyll cells from detached leaves (C). Average number of chloroplasts per cell is shown in (D) (n = 50, mean ± SE).
(E) Analysis of mesophyll cell size of wild-type and sbp mutant fully expanded leaves (n = 50, mean ± SE).
(F) Cell numbers counted from the large primary vein (midvein) to the leaf blade edge of the fully expended fifth leaves of wild-type and sbp mutant (n = 3 for wild-type and 6 for sbp, mean ± SE). Bars indicate 10 μm in (B) and 50 μm in (C). Error bars indicate standard errors (SE).
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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10. Figure 9
Starch Accumulation in Leaves of Wild-Type and sbp Mutant Plants.
(A) Images of wild-type and sbp mutant leaves stained with iodine/potassium iodide, an indicator of starch accumulation. Three-week-old wild-type and sbp mutant seedlings were harvested for staining.
(B) Analysis of starch contents in leaves of wild-type and sbp mutant plants (n = 3, mean ± SE). Error bars indicate standard errors (SE).
(C) TEM examination of starch granules in wild-type and sbp mutant chloroplasts.
Bars = 1 μm.
Molecular Plant 2012 5, DOI: ( /mp/sss012)
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11. Figure 10
Analysis of Sucrose-Dependent Growth and Development in Wild-Type and sbp Mutant Plants.
(A) Phenotypes of 35-day-old plants grown directly in peat soils (left panel) or the plants grown on half-strength MS medium with sucrose for 10 d, then transferred into peat soils and grown for 25 d (right panel).
(B) Phenotypic comparison of root growth of wild-type and sbp mutant seedlings grown vertically on half-strength MS medium with sucrose (upper panel) or without sucrose (lower panel).
(C) Phenotypic comparison of shoot growth of wild-type and sbp mutant seedlings grown on half-strength MS medium with sucrose or without sucrose.
(D) Analysis of primary root growth of wild-type and sbp mutant seedlings shown in (B) (n = 36, mean ± SE).
(E) Analysis of shoot growth of wild-type and sbp mutant seedlings shown in (C) (n = 36, mean ± SE). Error bars indicate standard errors (SE).
Molecular Plant 2012 5, DOI: ( /mp/sss012)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions