A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry

Abstract

It remains unclear whether the phloem unloading pathway alters to adapt to developmental transition in fleshy fruits that accumulate high level of soluble sugars. Using a combination of electron microscopy, transport of the phloem-mobile symplasmic tracer carboxyfluorescein, movement of the companion cell-expressed and the green fluorescent protein-tagged viral movement protein, and assays of the sucrose cleavage enzymes, the pathway of phloem unloading was studied in the berries of a hybrid grape (Vitis vinifera x Vitis labrusca). Structural investigations showed that the sieve element-companion cell complex is apparently symplasmically connected through plasmodesmata with surrounding parenchyma cells throughout fruit development, though a small portion of plasmodesmata are apparently blocked in the ripening stage. Both carboxyfluorescein and the green fluorescent protein-tagged viral movement protein were released from the functional phloem strands during the early and middle stages of fruit development, whereas the two symplasmic tracers were confined to the phloem strands during the late stage. This reveals a shift of phloem unloading from symplasmic to apoplasmic pathway during fruit development. The turning point of the phloem unloading pathways was further shown to be at or just before onset of ripening, an important developmental checkpoint of grape berry. In addition, the levels of both the expression and activities of cell wall acid invertase increased around the onset of ripening and reached a high level in the late stage, providing further evidence for an operation of the apoplasmic unloading pathway after onset of ripening. These data demonstrate clearly the occurrence of an adaptive shift of phloem unloading pathway to developmental transition from growing phase to ripening in grape berry.

Figure 1.

Structure of grape berry. A, Longitudinal section of a grape berry, showing the network of the vascular bundles, the loading site of CFDA into the axis of the cluster or the pedicel (indicated by red arrows), the sampling sites for structural observations, immunolabeling of acid invertases, and the CF transport imaging shown in Figure 2. B, Anatomical section of pericarp and mesocarp of grape berry. Arrows indicate the peripheral carpellary bundles. C, A transverse section of the phloem in the peripheral carpellary bundle at the beginning of developmental Stage I (10 d after anthesis). Arrows indicate plasmodesmata between CC and PC. D, A transverse section of the phloem in the peripheral carpellary bundle at the beginning of developmental Stage III. Arrows indicate plasmodesmata between SE and PC. E, SE-CC complexes in the peripheral carpellary bundle, showing two different classes of plasmodesmata between SE and CC (boxed-in areas), one being apparently normal and with branched channels as shown more clearly in the blow-up (F; arrows indicate the branched channels), and the other blocked by the electron-opaque globules (blow-up shown in G). H, A picture showing the electron-opaque globule-blocked plasmodesmata (arrows) between two PCs. Berries for ultrastructural observations in E to H were sampled at the beginning of developmental Stage III (see Fig. 2). Bars = 1 μm in C to E, and H. Abbreviations: cc, companion cell; pc, parenchyma cell; se, sieve element.

Figure 2.

CLSM imaging of CF (A–I) and 3a MP:GFP (J–P) unloading during development of grape berry. The sampling time is indicated in R. CF reached the berry phloem 3 to 4 h after loading of the pedicel or axis of the cluster (see Fig. 1A). The treated berry was sampled 72 h after CF loading and the sections by free hand or freezing microtome were prepared from the peripheral carpellary bundle zone (indicated in Fig. 1A). The red strands in A, D, and G and red area in B indicate the xylem zones labeled by Texas Red dextran. A to C, Berries collected in the middle of Stage I (around 30 d after anthesis). The green fluorescence of CF was shown to be released from the phloem strands (indicated by arrows) in longitudinal (A) and transverse (B) sections of the zones of the major peripheral carpellary bundle. C, Distribution of CF in PCs (a section by freezing microtome). D, Berries collected in the middle of Stage II (around 55 d after anthesis), showing release of CF from the phloem strands (indicated by arrow) of the branched minor peripheral carpellary bundle (see Fig. 1A). E and F, Berries collected in late Stage II (around 65 d after anthesis or 4–6 d before the onset of ripening). CF was shown to be released from phloem (indicated by arrows) in a longitudinal section of the major peripheral carpellary bundle (E) and to spread in PCs (F, a section by freezing microtome). G to I, Berries collected at the early (about 6 d after the onset of ripening; G) and the middle of Stage III (about 20 d after the onset of ripening; H and I). CF was confined to the phloem zones of the peripheral carpellary bundles in longitudinal (G and H, with arrows indicating phloem strands in G) and transverse (I) sections. J to L, Berries collected in the middle of Stage I (around 30 d after anthesis). J, The expression of 3a MP:GFP was clearly observed in CCs (indicated by red arrows) of the phloem of a minor peripheral carpellary bundle 15 h after the coincubation of berry tissue with CoYMV:3a MP:GFP-carrying Agrobacterium tumefaciens and began to spread to the surrounding cells (J, a section by freezing microtome). K, Release of 3a MP:GFP from the phloem zone (indicated by arrows) in a major peripheral carpellary bundle 20 h after the coincubation. L, Distribution of 3a MP:GFP in the PCs (a section by freezing microtome). M, Berries collected in the late Stage II (around 65 d after anthesis or 4–6 d before the onset of ripening). 3a MP:GFP spread from the phloem (indicated by arrow) of a minor peripheral carpellary bundle to surrounding PCs (a section by freezing microtome). “x” denotes xylem zone. N to P, Berries collected at the early (about 6 d after the onset of ripening, N) and the middle Stage III (about 20 d after the onset of ripening, O and P), showing that 3a MP:GFP was confined to the phloem zones of the peripheral carpellary bundles. N, A transverse section of a peripheral vascular strand. Phloem zone (p) is outlined by dotted yellow line and xylem (x) by dotted red line. Weak autofluorescence of xylem vessels can be seen in the xylem zone. O and P, Longitudinal sections of the major peripheral carpellary bundles. Q, The dimeric GFP fusion protein was expressed in and confined to CCs in phloem. R, A schema showing the developmental stages of grape berry and sampling time (the underlined letters A–P above the schema) in the above-described experiments. Berry growth (in volume) is expressed in relative data (%). The developmental Stage I represents the first rapid growth phase, Stage II the lag phase of growth, and Stage III the second rapid growth phase or ripening phase. The red arrow denotes the transition from Stage II to Stage III, i.e. onset of ripening. S, Time course of ¹⁴C-Suc transport from the loading sites in the pedicel phloem to berry tissues. ¹⁴C-Suc was fed to the pedicel in the same manner as CF was, and the ¹⁴C-Suc distribution in berries was detected by ¹⁴C-autoradiography at the indicated times (4, 6, 10, and 72 h after the ¹⁴C-Suc loading). Red circles indicate berry outlines. Bars = 100 μm in A to C, E, G, H, I, K, O, and P. Bars = 10 μm in D, F, J, L to N, and Q. Each value presented in R for soluble sugar concentrations is the mean of five replicates ± sd.

Figure 3.

CF transport imaging in whole berry around the onset of ripening and ¹⁴C-labeling of vascular bundles. A, For better guiding the observations of CF and ¹⁴C-assimilate transport, the vascular bundles were labeled by allowing berry to transpire safranine dye. A view at the pedicel side (a) and another view at the berry top side (b) show the network of the peripheral carpellary bundle (PB) and the position of the central carpellary bundle (CB). B, CF transport in whole berry. The red lines marked on berry models indicate the sectioning sites for the corresponding columns of the panels below the models. Berries were allowed to transport CF for 72 h, and then were collected and sectioned by free hand for observations under a fluorescence microscope. B, a to d, Berries treated by CFDA 6 d before onset of ripening. CF spread all over the berry from both the peripheral and central carpellary bundles (see A above and Fig. 1A for the network of the bundles). B, e to h, Berries treated by CFDA 1 d after onset of ripening. CF apparently spread, but to a much more reduced extent than before onset of ripening (see a–d). B, i to l, Berries treated by CFDA 4 d after onset of ripening. CF was restricted to both the peripheral and central carpellary bundles. C, ¹⁴C autoradiography in the middle of Stage II. ¹⁴C-labeled assimilates were transported in both the peripheral and central carpellary bundles, which are shown in longitudinal (a) and transverse (b) sections. Red circles in B and C indicate the outlines of the grape berry.

Figure 4.

Changes in the acid invertase amounts and activities and Suc synthase activities as well as soluble sugar concentrations during berry development. The amounts of acid invertases were assessed by immunoblotting (A and B) and by immunogold labeling (C–F) with the antisera directed against apple fruit acid invertases. A, For cell wall acid invertase, the amounts (CWI, top) and activities (white circles, middle) increased slowly from the early stage and maintained at a low level during the middle stage, and then increased rapidly around the onset of ripening and reached a high level in the late stage. The amounts (SAI, top) and activities (black circles, middle) of SAI changed substantially in an inverse pattern compared to cell wall invertase. Arrows in the middle section indicate the onset of ripening. The bottom section in A displays the changes in the Suc synthase activities in the cleavage direction (white triangles), the concentrations of Fru (black circles), Glc (white circles), and Suc (black triangles) in berry flesh, and total soluble sugars in the phloem exudates (black asterisks) and in the apoplasmic space of berries (black double triangles). B, The onset of ripening was shown to be the turning point of the increase of cell wall invertase amounts (CWI, section above) and activities (white circles, section below). The amounts (SAI, section above) and activities (black circles, section below) of SAI substantially changed inversely compared to cell wall invertase. The day “0” denotes the date of the onset of ripening and negative (−) numbers indicate the days before the onset of ripening, and the days after the onset of ripening are indicated by numbers 1 to 5. Arrows indicate the corresponding sampling time (at 4 and 2 d before the onset of ripening) of the columns of the immunoblotting data. C and D, Berries collected 6 d before the onset of ripening. C, Acid invertases visualized by immunogold particles were localized on cell walls between SE and CC and in the vacuole of CC. D, Acid invertases were shown to be numerous in the vacuole of PC with a lower density of the enzyme molecules on the cell wall between PCs. E and F, Berries collected 2 d after onset of ripening. E, Acid invertases on cell walls between SE and CC were shown to be much more numerous than those localized in the vacuole of CC. F, Acid invertases were predominantly localized on cell walls of PCs. Few acid invertase molecules were seen in vacuoles of PC. Bars = 1 μm. Abbreviations: cc, companion cell; cw, cell wall; pc, parenchyma cell; se, sieve element; v, vacuole. Each value presented in A and B is the mean of five replicates ± sd.