1. Tomato Ovary-to-Fruit Transition is Characterized by a Spatial Shift of mRNAs for Cell Wall Invertase and its Inhibitor with the Encoded Proteins Localized to Sieve Elements 
William M. Palmer, Lei Ru, Ye Jin, John W. Patrick, Yong-Ling Ruan 
Molecular Plant 
Volume 8, Issue 2, Pages (February 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
In Situ Hybridization of CWIN LIN5 mRNA in Tomato 2 DBA Ovaries and 2 DAA Fruitlets.
(A) A longitudinal section of a 2 DBA ovary hybridized with a LIN5 antisense probe. Note the dispersed pattern of LIN5 mRNA in columella (c), placenta (pl), and ovules (o), and in the ovary base adjoining the receptacle (r) in comparison with the sense control (B). Blue arrows point to the LIN5 mRNA detected by the antisense probe. ow, ovary wall.
(B) A consecutive section of (A) hybridized with a LIN5 sense probe as a negative control.
(C) A longitudinal section of a 2 DAA fruitlet hybridized with a LIN5 antisense probe. Note that the mRNA signal was restricted to the vasculatures of the pericarp (p), placenta (pl), and columella (c), highlighted by white, red, and yellow arrows, respectively. r, receptacle; s, seed.
(D) A consecutive section of (C) hybridized with a LIN5 sense probe as a negative control. Scale bar represents 250 μm.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2
In Situ Hybridization of INH1 mRNA in Tomato 2 DBA Ovaries and 2 DAA Fruitlets.
(A) A longitudinal section of a 2 DBA ovary hybridized with an INH1 antisense probe. Note the dispersed pattern of INH1 mRNA in columella (c), placenta (pl), ovules (o), ovary wall (ow), and receptacle (r) in comparison with the sense control (B). Blue arrows point to the INH1 mRNA detected by the antisense probe.
(B) A consecutive section of (A) hybridized with an INH1 sense probe.
(C) A longitudinal section of a 2 DAA fruitlet hybridized with an INH1 antisense probe. Note that the INH1 mRNA became restricted to the vasculature of the pericarp (p), placenta (pl), and columella (c), highlighted by white, red, and yellow arrows, respectively.
(D) A consecutive section of (C) hybridized with an INH1 sense probe. Scale bar represents 250 μm.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3
Immunolocalization of LIN5 and INH1 in Vasculatures of 2 DBA Ovaries and Surrounding Tissues.
(A) A longitudinal section of a flower stained with Toluidine blue depicting anatomical structures. c, columella; o, ovule; ow, ovary wall; pet, petal; pl, placenta; r, receptacle; sta, stamen.
(B) A consecutive section of (A) immunolabeled with LIN5 antiserum. Red arrowheads indicate immunolocalized LIN5 located throughout the flower vasculature.
(C) A consecutive section of (B) immunolabeled with INH1 antiserum. Red arrows indicate immunolocalized INH1 located throughout the flower vasculature. Each image of (B) and (C) was a montage of 70 images acquired under a 20× objective, then stitched together using Adobe Bridge and Adobe Photoshop CS4 software packages. Red and yellow boxes indicate areas for subsequent immunolocalization in Figures 4 and 5 to ascertain cell-specific localization of LIN5 and INH1, respectively. Scale bar represents 350 μm. Negative control with pre-immune serum exhibited no signal (data not shown).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4
LIN5 Was Immunologically Localized to Cell Walls of Sieve Elements of 2 DBA Tomato Ovaries.
(A) A section from the placental vascular region of a 2 DBA ovary (see Figure 3B) labeled with LIN5 antiserum. Note the florescent signals from a subset of distinctive cells.
(B) The same section as (A), counterstained with Aniline blue for callose, revealed fluorescence of Aniline blue bound to callose at specific cellular sites.
(C) The same section counterstained with DAPI detecting nuclei in cells surrounding those immunolocalized with LIN5 antibody (see A).
(D) The same section counterstained for cell wall cellulose with Calcofluor white.
(E) Overlay of the four images from (A) to (D) to identify cells displaying LIN5 immunolocalization. Cells exhibiting LIN5 signals contained callose but were enucleate, a characteristic of sieve elements (SE). Note that the LIN5 was immunolocalized to walls of SEs exhibiting Calcofluor white fluorescence (red color in the magnified circled inset), consistent with LIN5 being a cell wall protein. Also note the absence of LIN5 and callose from, but the presence of nuclei in, the adjacent companion cells (CC) and phloem parenchyma (PP). Scale bar represents 10 μm. SP, sieve plate. Negative control with pre-immune serum exhibited no LIN5 signal (see Supplemental Figure 1).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

6. Figure 5
INH1 Was Immunologically Localized to Sieve Elements in 2 DBA Tomato Ovaries.
(A) A section from the placental vascular region of 2 DBA ovary (see Figure 3C) was labeled with INH1 antiserum, which led to florescent signals arising from a subset of distinctive cells.
(B) The same section of (A) was counterstained with Aniline blue for callose, which revealed signals in specific cellular sites.
(C) Further counterstaining of the same section with DAPI for nuclei in the surrounding cells of those exhibiting INH1 signals in (A).
(D) Finally, the section was counterstained with Calcofluor white for cell wall cellulose.
(E) Overlay of the four images from (A) to (D) to identify the cells displaying INH1, demonstrating that the cells exhibiting INH1 signals contained callose but were enucleated, characteristic of mature sieve elements (SE). Note that the signal was localized in the SE cell walls exhibiting Calcofluor white, consistent with INH1 being a cell wall protein. Also note the absence of INH1 and callose but the presence of nuclei in the adjacent companion cells (CC) and phloem parenchyma (PP). Scale bar represents 10 μm. Negative control with pre-immune serum exhibited no INH1 signal (see Supplemental Figure 2).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

7. Figure 6
LIN5 and INH1 Were Immunologically Localized to Sieve Elements of 2 DAA Tomato Fruitlets.
(A) A section from the placental vascular region of a 2 DAA fruitlet, sequentially labeled with Lin5 antiserum, Aniline blue, DAPI, and Calcofluor white as described for Figures 4 and 5.
(B) A consecutive section of (A), sequentially labeled with INH1 antiserum, Aniline blue, DAPI, and Calcofluor white. Note that the cells exhibiting green fluorescent signals (green arrows) for LIN5 in (A) and INH1 in (B) contained Aniline blue-labeled callose in sieve plates (white arrows) but were enucleate, a characteristic of sieve elements (SE). Scale bar represents 10 μm.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

8. Figure 7
LIN5 Was Expressed in Sieve Elements but Not in Provascular Cells of Flower Buds at 15 DBA.
(A) Bright-field image of a resin-embedded section of a flower bud at 15 DBA stained with Toluidine blue. Scale bar represents 200 μm.
(B) Zoomed view of the boxed region in (A) showing a petal section with differentiated vascular bundles (green box) and provascular bundles (PVB, yellow box) Scale bar represents 65 μm.
(C) Bright-field and fluorescent images of a vascular bundle (VB). The VB highlighted in C1 displayed immunologically labeled LIN5 (FITC fluorescence) in some cells (arrows in C2). Note that the cells immunolabeled with LIN5 were enucleate, as indicated by the absence of any DAPI staining (arrows in C3), but exhibited callose as indicated by Aniline blue counterstaining (arrows in C4). These observations show that LIN5 was immunolocalized exclusively to sieve elements.
(D) Bright-field and fluorescent images of a provascular bundle. The provascular bundle highlighted in D1 displayed no immunolabeling of LIN5 (D2). Note that the provascular bundle contained elongated nuclei, indicated by the blue fluorescence by counterstaining with DAPI (arrows in D3), but no callose detectable by Aniline blue counterstaining (D4). These data show that, in contrast to that in sieve elements (C1–C4), LIN5 was not expressed in provascular bundles. Scale bars represent 10 μm in C1–C4 and D1–D4.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

9. Figure 8
Western Blot Analyses of LIN5 and INH1 and Enzyme Activity Assays in Extracts from Ovaries and Young Fruitlets during Fruit Set.
(A) Coomassie blue staining of cell wall (CWP) and total soluble proteins (TSP) extracted from ovaries at 2 DBA and at anthesis (0 DAA) and fruitlets at 2 DAA. Each lane contains 10 μg of protein.
(B) The protein gel blot probed with LIN5 antiserum detected a ∼64 kDa band corresponding to the predicted size of LIN5 in all the cell wall fractions but not in the soluble fractions.
(C) The same blot in (B) was stripped and re-probed with INH1 antiserum. A ∼16 kDa band corresponded to the predicted size of INH1 in all the cell wall fractions but not in the soluble fractions.
(D) CWIN activity in ovaries and fruitlets at 2 DBA, 0 DAA, and 2 DAA.
(E) VIN and CIN activities in ovaries and fruitlets at 2 DBA, 0 DAA, and 2 DAA. Each value in (D) and (E) is the SE ± mean of at least three biological replicates. Different letters in (D) and (E) indicate significant difference at P < and P < 0.5, respectively, according to randomized one-way ANOVA test.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

10. Figure 9
A Defective CWIN, SldeCWIN1, Expressed in 2 DBA Tomato Ovaries but Barely Detectable in 2 DAA Fruitlets.
(A) Partial multiple sequence alignment of amino acid sequences of AtCWIN1 (AY079422), SlCWIN1 (NM_ ), NtNin88 (AF376773), and SldeCWIN1 (XM_ ). Two conserved domains are magnified: (I) β-fructosidase domain; (II) catalytic domain. Two crucial amino acids (Trp47 and Asp239) required for sucrose hydrolysis are underlined in bold. Note that SldeCWIN1 has mutations in both amino acids, characteristic of defective CWINs as shown for NtNin88 in tobacco (see Le Roy et al., 2013).
(B) Transcript levels of SldeCWIN1, SIINH1, and SlCWIN1 measured by quantitative PCR in 2 DBA ovaries and 2 DAA fruitlets. Each bar represents mean ± SE of four biological replicates.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

11. Figure 10
Phloem Unloading Follows an Apoplasmic Pathway in 2 DBA Ovary and a Symplasmic Pathway in 2 DAA Fruit Pericarp as Revealed by Monitoring Movement of a Symplasmic Dye, CF.
(A) CF fluorescence detected in the ovary pedicel after 24 h feeding CFDA. Note that much more CF fluorescence appeared in the inner (ip) than in the external phloem (ep). Also note the auto-fluorescence of the xylem (x) as shown in the inset.
(B) No CF fluorescence was detectable in a 2 DBA ovary after feeding CFDA for 24 h.
(C) The same section as (B), but viewed under bright field to show the structure of the ovary including the ovary wall (ow), placenta (pl), and ovule (o).
(D) CF fluorescence was detected in the phloem of ovary wall (ow) after feeding CFDA for 72 h. CF was confined to phloem of primary vascular bundles (pv, arrows) and did not spread laterally to the surrounding cells. CF fluorescence was also detected in the placenta (pl) but not in the ovules (o).
(E) CF fluorescence detected in the pedicel of 2 DAA fruitlets after 24 h feeding CFDA. Most CF fluorescence appeared in the internal phloem (ip), with xylem (x) exhibiting blue auto-fluorescence (see inset).
(F) CF fluorescence appeared in phloem of 2 DAA fruit pericarp (p) after 24 h feeding CFDA (arrowheads) in primary (pv) and secondary (sv) vascular bundles. The inset from the boxed area showed CF in phloem (arrows) but not in the xylem (x). The latter displayed blue auto-fluorescence. CF also spread to a large part of the placenta (pl), but not to seeds (s).
(G) The same section as (F), but viewed under bright field to show the structure of the fruit including the location of the seeds (s).
(H) CF fluorescence moved to cells beyond the phloem (circle) in the pericarp (p) of 2 DAA fruitlets after feeding CFDA for 72 h. CF fluorescence spread throughout the placenta (pl), but did not move to the seed (s). Scale bar represents 100 μm in (A). The remaining images have the same magnification as (A).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions