Ricinosomes predict programmed cell death leading to anther dehiscence in tomato

Abstract

Successful development and dehiscence of the anther and release of pollen are dependent upon the programmed cell death (PCD) of the tapetum and other sporophytic tissues. Ultrastructural examination of the developing and dehiscing anther of tomato (Solanum lycopersicum) revealed that cells of the interlocular septum, the connective tissue, the middle layer/endothecium, and the epidermal cells surrounding the stomium all exhibit features consistent with progression through PCD. Ricinosomes, a subset of precursor protease vesicles that are unique to some incidents of plant PCD, were also present in all of these cell types. These novel organelles are known to harbor KDEL-tailed cysteine proteinases that act in the final stages of corpse processing following cell death. Indeed, a tomato KDEL-tailed cysteine proteinase, SlCysEP, was identified and its gene was cloned, sequenced, and characterized. SlCysEP transcript and protein were restricted to the anthers of the senescing tomato flower. Present in the interlocular septum and in the epidermal cells surrounding the stomium relatively early in development, SlCysEP accumulates later in the sporophytic tissues surrounding the locules as dehiscence ensues. At the ultrastuctural level, immunogold labeling localized SlCysEP to the ricinosomes within the cells of these tissues, but not in the tapetum. It is suggested that the accumulation of SlCysEP and the appearance of ricinosomes act as very early predictors of cell death in the tomato anther.

Figure 1.

Structure and development of the tomato androecium and anther. A, Whorl of anthers constituting the androecium. Bar = 200 μm. B, Tissues/structures of the anther. c, Connective tissue; e, epidermis; en, endothecium; is, interlocular septum; l, locule; m, middle layer; st, stomium; t, tapetum; v, vasculature. Bar = 100 μm. C to G, Developmental series illustrating the progressive loss of sporophytic cell layers/structures leading up to dehiscence. Tapetal layers apparent at stage 9 (C) and 11 (D) begin to be lost by stage 13 (E), the interlocular septum being close to disruption at that time (E), and loss of the septum is seen by stage 16 (F). There is a progressive loss of cell layers of the connective tissue and the middle layer/endothecium from stage 13 (E) through stage 16 (F) leading to dehiscence (G), with the cells surrounding the stomium separating at stage 20 (G), marking the completion of the dehiscence process. Bar in G = 100 μm. [See online article for color version of this figure.]

Figure 2.

Stomial cells of the developing tomato anther display features consistent with the progression of PCD. A to F, Developmental series roughly equivalent to that seen in Figure 1, C to G. Stages 9 (A), 11 (B), 15 (C and D), 18 (E), and near 20 (F) are shown. A, Stage 9 epidermal cells surrounding the stomium appear normal with the exception of the presence of ricinosomes (stars). Cellular material in the central vacuole suggests that autophagy is occurring. The cell illustrated has recently divided, with new cell wall forming between adjacent sibling cells (cw, with white arrow). Ricinosomes are also apparent in the interlocular septal cell lying immediately beneath the epidermal cells (bottom). B, Stage 11 epidermal cell showing the accumulation of autophagic vesicles/vacuoles (av) in the cytoplasm. Ricinosomes are evident (stars) in the stomial cells. Features of the cytoplasm of the interlocular septal cell (bottom right) are difficult to discern. C and D, Stage 15. As development and progression to dehiscence continue, the plasma membrane of the stomial cells pulls away from the cell wall. Autophagy remains evident (C and D). Ricinosomes may be more numerous, and the cytoplasm becomes somewhat flocculent in appearance (D). The interlocular septal cell is dead by this stage (bottom right in C), evidenced by the loss of cytoplasm (see Fig. 1E). E, By stage 18, the cytoplasm of the stomial cells has condensed and nuclei are somewhat misshapen, with ultrastuctural details of the cytoplasm being difficult to discern. Cytoplasm is retracted from the cell wall, and ricinosomes remain. F, Upon death, the central vacuole and cytoplasm collapse and recognizable cytoplasmic structures are lost. Ricinosomes are no longer apparent. G to J, Details of ricinosomes in the stomial cells. G and H, Stages 9 and 11, respectively. White arrowheads indicate potential continuation of the ricinosome and rough endoplasmic reticulum membranes. I, Stage 15. Ricinosome membranes are studded with ribosomes. J, Stage 18. Numerous ricinosomes present in the very condensed cytoplasm. er, (Rough) endoplasmic reticulum; m, mitochondria; n, nucleus; p, plastid; v, vacuole. Bars = 1 μm in A to F and 0.5 μm in G to J.

Figure 3.

Developmental series showing cells of the connective tissue and cells of the middle layer/endothecium. A to D, Connective tissue cells. Cells of the connective tissue display features consistent with the progression of PCD, including vesiculation of the cytoplasm (A, stage 11), shrinkage of the plasma membrane away from the cell wall (cw; B, stage 15; C, stage 18) with ricinosomes occasionally evident in the cytoplasm (B, stage 15), and finally vacuolar and cytoplasmic collapse near stage 20 (D). Nuclei within the connective tissue cells demonstrate some abnormalities in shape as development proceeds (A and C). Interlocular septal cells are seen at top (A) and top left (B and C), identified by the voids left from the removal of presumed calcium oxylate crystals during tissue processing. E to H, Middle layer/endothecium cells. Nuclear and mitochondrial aberrations and the occurrence of large peroxisomes are more apparent in these cells (E–G show stages 11, 15, and 18, respectively). Some retraction of the cytoplasm from the cell wall, vesiculation, and autophagy are apparent (G), until the final collapse of the central vacuole (H, near stage 20). Ricinosomes are not readily apparent in the cytoplasm until late in development (H). The locule would be located toward the bottom of the image in E to H, with PCD progressing outward from the locule through the cell layers. I to M, Ricinosomes are indeed present early in development (I–M show stages 11, 13, 15, 16, and near 20, respectively), but contrast is similar to the cytosol. Remains of the tapetum are seen on the left side of the image in L. Bars = 1 μm in A to H and 0.5 μm in I to M. av, Autophagic vesicles/vacuoles; er, (rough) endoplasmic reticulum; G, Golgi; m, mitochondria; n, nucleus; p, plastid; px, peroxisome; v, vacuole.

Figure 4.

Multiple alignment of tomato SlCysEP, castor CysEP, mung bean SH-EP, pea TPE4A, and Hemerocallis species SEN11, showing the ERFNIN and KDEL motifs and the catalytic residues. Predicted cleavage sites for the signal peptides and the prodomain are shown with arrows.

Figure 5.

Southern blot of digested tomato genomic DNA probed with cDNA complementary to exon 1 of SlCysEP. Lanes show genomic DNA digested with XbaI (1), HindIII (2), DraI (3), NcoI (4), and EcoRV (5). Note that the probe detects only one fragment of genomic DNA in each digest, suggesting that SlCysEP exists as a single-copy gene.

Figure 6.

Enzymatic properties of recombinant SlCysEP. A, pH optimum for rSlCysEP activity at 37°C using azocasein as substrate. Error bars represent the sd between triplicate absorbance values for each pH. B, Effect of DTT and E64 on proteolysis by rSlCysEP and human cathepsin B at optimal pH at 37°C. DTT (white bars) increases activity of both enzymes over that seen in control reaction (gray bars). E64 (black bars) completely abolishes activity. Error bars represent the sd between triplicate independent assays. C, Western-blot analysis of rSlCysEP self-hydrolysis in acidic pH. Self-hydrolysis occurs within 3 min at pH 4.8 but does not occur at pH 7.0. Values above each lane indicate reaction time in minutes. MW, Molecular mass.

Figure 7.

Accumulation of SlCysEP transcript and protein in developing tomato flowers. A, Northern-blot analysis using total flower RNA from whole flowers at different developmental stages and from floral tissues of stage 13 to 15 flower buds. 25S ribosomal RNA is shown as a loading control. B, Western blot of total flower protein from different developmental stages and different floral tissues of stage 13 to 15 flowers detected with affinity-purified anti-SlCysEP rabbit polyclonal antibodies. Pd, Pedicel; Se, sepal; Pe, petal; St, stamen; Ca, carpel.

Figure 8.

Immunohistochemical localization of SlCysEP in sporophytic structures/cell layers of the tomato anther leading up to dehiscence. A to D, SlCysEP is localized to the interlocular septal cells at stage 11 (A; blue/purple, inset), the connective tissue, middle layer/endothecium, and epidermal cells surrounding the stomium at stage 13 (B), and to the outer cell layer of the connective tissue and cells near the stomium at stages 16 (C) and 20 (D). E to H, Preimmune controls for A to D, respectively. Bar = 100 μm.

Figure 9.

Immunogold localization of SlCysEP in sporophytic tissues of tomato anther. A to D, Stomial cells. E to I, Cells of the connective tissue. J to M, Middle layer/endothecium cells. A, E, and J, Early development, approximately stages 9 to 11. B, F to H, and K to L, Mid to late development, approximately stages 15 to 18. C, D, I, and M, Late development/dehiscence following vacuolar collapse. Tapetum is seen on the right side of J. SlCysEP is localized to ricinosomes (stars) in all cases (A, B, E to H, and J to L) until the central vacuole collapses, after which SlCysEP is released to the cytosol (C, D, I, and M). The inset in C shows a control using anti-SlCysEP antibodies preabsorbed with rSlCysEP. Bars = 0.5 μm. cw, Cell wall; m, mitochondria; n, nucleus; p, plastid; v, vacuole.

Figure 10.

Transcript analysis of several Cys proteinase genes during anther development. Gene-specific primers were used to amplify regions of Cys proteinase transcripts via RT-PCR on total RNA from stages 1 to 13, 13 to 18, and 18 to 20 anthers. Actin is included as a loading control.