Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage

The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed.     

Phloem loading and unloading of sugars and amino acids

In terrestrial higher plants, phloem transport delivers most nutrients required for growth and storage processes. Some 90% of plant biomass, transported as sugars and amino nitrogen (N) compounds in a bulk flow of solution, is propelled though the phloem by osmotically generated hydrostatic pressure differences between source (net nutrient export) and sink (net nutrient import) ends of phloem paths. Source loading and sink unloading of sugars, amino N compounds and potassium largely account for phloem sap osmotic concentrations and hence pressure differences. A symplasmic component is characteristic of most loading and unloading pathways which, in some circumstances, may be interrupted by an apoplasmic step. Raffinose series sugars appear to be loaded symplasmically. However, sucrose, and probably certain amino acids, are loaded into minor veins from source leaf apoplasms by proton symporters localized to plasma membranes of their sieve element/companion cell (se/cc) complexes. Sucrose transporters, with complementary kinetic properties, are conceived to function as membrane transporter complexes that respond to alterations in source/sink balance. In contrast, symplasmic unloading is common for many sink types. Intervention of an apoplasmic step, distal from importing phloem, is reserved for special situations. Effluxers that release sucrose and amino acids to the surrounding apoplasm in phloem loading and unloading are yet to be cloned. The physiological behaviour of effluxers is consistent with facilitated membrane transport that can be energy coupled. Roles of sucrose and amino acid transporters in phloem unloading remain to be discovered along with mechanisms regulating symplasmic transport. The latter is hypothesized to exert significant control over phloem unloading and, in some circumstances, phloem loading.     

Acid invertase is predominantly localized to cell walls of both the practically symplasmically isolated sieve element/companion cell complex and parenchyma cells in developing apple fruits

The acid invertase (β‐fructosidase, EC 3·2·1·26) was localized at subcellular level via immunogold electron microscopy in the phloem‐unloading zone of developing apple fruit. The enzyme (immunogold particles) was found to reside predominantly in the cell walls of the sieve element/companion cell (SE/CC) complex, phloem parenchyma cells and other parenchyma cells. There was almost no gold particle found in cytoplasm and vacuole. This distribution pattern remained unchanged throughout the growing season, but the enzyme numbers varied. The density of immunogold particles increased during fruit development. The immunoblotting of soluble and insoluble acid invertases provided a supporting proof for the assays of immunolocalization. The biochemical analysis showed a predominantly cell‐wall‐distributed activity of acid invertase that corresponds essentially with its amount distribution. The ultrastructural observations showed that there were numerous plasmodesmata between the parenchyma cells, but almost no plasmodesmium between the SE/CC complex and its surrounding parenchyma cells, practically resulting in the symplasmic isolation of the SE/CC complex. It is therefore suggested that the unloading pathway of sucrose from the SE/CC complex may be predominantly apoplasmic in the developing apple fruit, and that the unloaded sucrose may be hydrolysed by the functional acid invertase localized in the cell wall before it is loaded in sink cells.     

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

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.     

Sucrose carrier RcSCR1 is involved in sucrose retrieval, but not in sucrose unloading in growing hypocotyls of Ricinus communis L

The transport of assimilates from source to sink tissues is mediated by the phloem. Along the vascular system the phloem changes its physiological function from loading phloem to transport and unloading phloem. Sucrose carrier proteins have been identified in the transport phloem, but it is unclear whether the physiological role of these transporters is phloem unloading of sucrose or retrieval of apoplasmic sucrose back into the sieve element/companion cell complex. Here, we describe the dynamic expression of the Ricinus communis sucrose carrier RcSCR1 in the hypocotyl at different sink strengths. Our results indicate that phloem unloading in castor bean is not catalysed by the phloem loader RcSCR1. However, this sucrose carrier represents the molecular basis of the sucrose retrieval mechanism along the transport phloem, which is dynamically adjusted to the sink strength. As a consequence, we assume that other release carrier(s) exist in sink tissues, such as the hypocotyl, in R. communis.     

Transporter SlSWEET15 unloads sucrose from phloem and seed coat for fruit and seed development in tomato

Tomato (Solanum lycopersium), an important fruit crop worldwide, requires efficient sugar allocation for fruit development. However, molecular mechanisms for sugar import to fruits remain poorly understood. Expression of sugars will eventually be exported transporters (SWEETs) proteins is closely linked to high fructose/glucose ratios in tomato fruits and may be involved in sugar allocation. Here, we discovered that SlSWEET15 is highly expressed in developing fruits compared to vegetative organs. In situ hybridization and β-glucuronidase fusion analyses revealed SlSWEET15 proteins accumulate in vascular tissues and seed coats, major sites of sucrose unloading in fruits. Localizing SlSWEET15-green fluorescent protein to the plasma membrane supported its putative role in apoplasmic sucrose unloading. The sucrose transport activity of SlSWEET15 was confirmed by complementary growth assays in a yeast (Saccharomyces cerevisiae) mutant. Elimination of SlSWEET15 function by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated protein gene editing significantly decreased average sizes and weights of fruits, with severe defects in seed filling and embryo development. Altogether, our studies suggest a role of SlSWEET15 in mediating sucrose efflux from the releasing phloem cells to the fruit apoplasm and subsequent import into storage parenchyma cells during fruit development. Furthermore, SlSWEET15-mediated sucrose efflux is likely required for sucrose unloading from the seed coat to the developing embryo.

SlSWEET15, a specific sucrose uniporter in tomato, mediates apoplasmic sucrose unloading from phloem cells and seed coat to support fruit expansion and seed filling.     

植物体内光合同化物韧皮部装载和卸出研究进展

近年来研究表明,植物体内光合同化物的韧皮部装载和卸出均有其本途径和质外体途径,装载转运的糖类主要有：（２）棉子糖及其人类似物（以共质体方式装载）;（２）蔗糖（以质外体方式装载）。同化物的共质体卸出可通过扩散和集中作用实现,而质外体卸出则根据蔗糖在质外体是否水解而分为两种类型。卸出和装载的途径、机理因植物种类及库源关系而不同,也会受生长发育阶段及环境的变化而调整。深入研究韧皮部装载和帛出调控机制,对     

Effects of low light on phloem ultrastructure and subcellular localization of sucrose synthase in <Emphasis Type="Italic">Prunus persica</Emphasis> L. var. <Emphasis Type="Italic">nectarina</Emphasis> Ait. fruit

Using electron microscopy, the ultrastructure of phloem unloading zone was examined in the Prunus persica L. var. nectarina Ait. fruit. Our study showed that, in the SE/CC (sieve element/companion cell) complexes, CC developing under low light had a thin cytoplasm layer with few mitochondria and numerous small vacuoles, and not clearly seen nuclei. The cytoplasm vacuolation indicated that the cytoskeleton was destroyed at low light. The effects of low light on CC development suggest that unloading evidently linked to the low accumulation of soluble sugars by fruit. At the young fruit stage, flesh parenchyma around the phloem tissue had no starch grains in the plastids in fruit developing under low light. This is a further indication that less photoassimilates was translocated from source leaves to fruit sinks under low light during the young fruit developmental stage. The activity of sucrose synthase (SuSy), the key enzyme of sucrose metabolism in fruit, increased dramatically during fruit maturation. The highest SuSy activity during the rapid fruit growth phase suggests that sink strength could be correlated with the SuSy activity. The high SuSy activity under normal light possibly indicates that fruit had a capacity to utilize sucrose irrespective of their site of phloem unloading. Immunogold electron microscopy showed that SuSy was localized mainly in the vacuole of flesh parenchyma cells. The vacuole-localized SuSy can hydrolyze sucrose imported from the phloem, which may explain the apparent correlation between SuSy activity and phloem unloading. The double sieve element (SE/SE) complexes occurred in a greater number and had thicker cell walls under normal light intensity than under low light intensity. These data demonstrate clearly that low light decreased SuSy activity in the control of phloem unloading. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 509–517. This text was submitted by the authors in English.     

Evidence for apoplasmic phloem unloading in developing apple fruit

The phloem unloading pathway remains unclear in fleshy fruits accumulating a high level of soluble sugars. A structural investigation in apple fruit (Malus domestica Borkh. cv Golden Delicious) showed that the sieve element-companion cell complex of the sepal bundles feeding the fruit flesh is symplasmically isolated over fruit development. 14C-autoradiography indicated that the phloem of the sepal bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the sepal bundles from the basal to the apical region of the fruit. A 52-kD putative monosaccharide transporter was immunolocalized predominantly in the plasma membrane of both the sieve elements and parenchyma cells and its amount increased during fruit development. A 90-kD plasma membrane H(+)-ATPase was also localized in the plasma membrane of the sieve element-companion cell complex. Studies of [14C]sorbitol unloading suggested that an energy-driven monosaccharide transporter may be functional in phloem unloading. These data provide clear evidence for an apoplasmic phloem unloading pathway in apple fruit and give information on the structural and molecular features involved in this process.     

Localization of ATPase in Sink Tissues of Ricinus

Histochemical localization of ATPase was carried out on phloemtissues from vegetative and reproductive sinks of Ricinus communis,using lead precipitation procedures. Reaction products werelocalized mainly at the plasma membrane of the sieve elements,companion cells and phloem parenchyma cells. Activity was alsopresent in plasmodesmata, the tonoplast of companion cells anddispersed P-protein within the sieve element lumen. The resultsare discussed in relation to the possible involvement of a plasmamembrane ATPase in apoplastic and symplastic unloading fromthe phloem conducting tissues. ATPase, sink tissues, unloading, Ricinus communis     

Dynamic characteristics of sugar accumulation and related enzyme activities in sweet and non-sweet watermelon fruits

Sugar content largely determines watermelon fruit quality. We compared changes in sugar accumulation and activities of carbohydrate enzymes in the flesh (central portion) and mesocarp of elite sweet watermelon line 97103 (Citrullus lanatus subsp. vulgaris) and exotic non-sweet line PI296341-FR (C. lanatus subsp. lanatus) to elucidate the physiological and biochemical mechanisms of sugar accumulation in watermelon fruit. The major translocated sugars, raffinose and stachyose, were more unloaded into sweet watermelon fruit than non-sweet fruit. During the fruit development, acid α-galactosidase activity was much higher in flesh of 97103 than in mesocarp of 97103, in flesh and mesocarp of PI296341-FR fruit. Insoluble acid invertase activity was higher in 97103 flesh than in 97103 mesocarp, PI296341-FR flesh or mesocarp from 18 days after pollination (DAP) to 34 DAP. Changes in soluble acid invertase activity in 97103 flesh were similar to those in PI296341-FR flesh and mesocarp from 18 DAP to full ripening. Sucrose synthase and sucrose phosphate synthase activities in 97103 flesh were significantly higher than those in 97103 mesocarp and PI296341-FR fruits from 18 to 34 DAP. Only insoluble acid invertase and sucrose phosphate synthase activities were significantly positively correlated with sucrose content in 97103 flesh. Therefore, phloem loading, distribution and metabolism of major translocated sugars, which are controlled by key sugar metabolism enzymes, determine fruit sugar accumulation in sweet and non-sweet watermelon and reflect the distribution diversity of translocated sugars between subspecies.     

Increased Expression of a Phloem Membrane Protein Encoded by NHL26 Alters Phloem Export and Sugar Partitioning in Arabidopsis

The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield. Similar effects were observed when NHL26 was overexpressed in companion cells, under the control of a companion cell–specific promoter. The soluble sugar content of the phloem sap and sink organs was lower than that in the wild type, providing evidence of a sugar export defect. This was confirmed in a phloem-export assay with the symplastic tracer carboxyfluorescein diacetate. Leaf sugar accumulation was accompanied by higher organic acid, amino acid, and protein contents, whereas analysis of the metabolite profile of phloem sap exudate revealed no change in amino acid or organic acid content, indicating a specific effect on sugar export. NHL26 was found to be located in the phloem plasmodesmata and the endoplasmic reticulum. These findings reveal that NHL26 accumulation affects either the permeability of plasmodesmata or sugar signaling in companion cells, with a specific effect on sugar export.     

Phloem unloading in developing walnut fruit is symplasmic in the seed pericarp and apoplasmic in the fleshy pericarp

The sieve element-companion cell (SE-CC) complex of the sepal bundles feeding the fleshy pericarp of developing walnut (Juglans regia L.) fruit is structurally symplasmically isolated, but the SE-CC complex of the minor ventral carpellary bundles located in the seed pericarp and feeding the seed is structurally symplasmically connected to its adjacent parenchyma cells. 14C-autoradiography indicated that the phloem of both the sepal and carpellary bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye is confined to the phloem strands of the sepal bundles in the fleshy pericarp, but released from the phloem strands of the minor ventral carpellary bundles into the surrounding parenchyma cells in the seed pericarp. A 60-kDa acid invertase was immunolocalized to the cell wall of SE-CC complex and parenchyma cells in both the fleshy and seed pericarp. These data provide clear evidence for an apoplasmic phloem unloading pathway in the fleshy pericarp and a predominant symplasmic phloem unloading pathway parallel with a possible apoplasmic path as suggested by the presence of the extracellular invertase in the seed pericarp. A model of complex phloem unloading pathways in developing walnut fruit has been proposed.     

A dual switch in phloem unloading during ovule development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Developing flowers are important sinks in Arabidopsis thaliana. Their energy demand is covered by assimilates which are synthesized in source leaves and transported via the vasculature. Assimilates are unloaded either symplastically through plasmodesmata or apoplastically by specific transport proteins. Here we studied the pathway of phloem unloading and post-phloem transport in developing gynoecia. Using phloem-mobile fluorescent tracers, we show that phloem unloading into cells of ovule primordia followed a symplastic pathway. Subsequently, the same tracers could not move out of phloem cells into mature ovules anymore. A further change in the mode of phloem unloading occurred after anthesis. In open flowers as well as in outgrowing siliques, the phloem was again unloaded via the symplast. This observed onset of symplastic phloem unloading was accompanied by a change in frequency of MP17-GFP-labeled plasmodesmata. We could also show that the change in cell–cell connectivity was independent of fertilization and increasing sink demand. The presented results indicate that symplastic connectivity is highly regulated and varies not only between different sink tissues but also between different developmental stages.     

Abscisic acid activates ATPase in developing apple fruit especially in fruit phloem cells

Abscisic acid (ABA) improves the sink strength by promoting the phloem unloading and regulating the assimilate metabolism in the economic sink organs of crops, although its mechanism remains unknown. The present experiment, using the techniques of the in vivo injection of ABA into the intact apple fruit attached to a growing apple tree and the in vivo incubation of the fruit tissue in the ABA‐contained medium, showed that ABA strongly activated the fruit ATPase especially P‐ATPase, of which the activity was doubled by ABA treatment. This ATPase activation was shown to be in vivo tissue‐dependent. The ABA‐induced P‐ATPase activation was fruit developmental stage‐, ABA dose‐, medium pH‐ and incubation time‐dependent. Physiological active (+)ABA was shown more effective to stimulate P‐ATPase activity than (+/–)ABA, and two ABA analogues (–)ABA and trans‐ABA, had no effect on P‐ATPase activation, indicating that only physiologically active cis(+)ABA can induce the enzyme activation, and so the ABA‐induced effects are stereospecific. The protein synthesis inhibitor cycloheximide was shown to have no effect on P‐ATPase activation by ABA, suggesting that synthesis of new proteins was not involved in the enzyme activation. The cytochemical assay revealed that P‐ATPase was activated by ABA in both the phloem and its surrounding flesh parenchyma cells, and that the most strongly P‐ATPase activation was observed in the plasma membrane of sieve element/companion cell complex. These data suggest that the improvement of phloem unloading by ABA previously reported in this fruit as in other crop sink organs may be attributed, at least partly, to the ABA‐induced ATPase activation especially in phloem cells.     

Genotypic differences in pod wall and seed growth relate to invertase activities and assimilate transport pathways in asparagus bean

Background and Aims

Coordination of sugar transport and metabolism between developing seeds and their enclosing fruit tissues is little understood. In this study the physiological mechanism is examined using two genotypes of asparagus bean (Vigna unguiculata ssp. sesquipedialis) differing in pod wall and seed growth rates. Pod growth dominates over seed growth in genotype ‘Zhijiang 121’ but not in ‘Zhijiang 282’ in which a ‘bulging pod’ phenotype is apparent from 8 d post-anthesis (dpa) onward.

Methods

Seed and pod wall growth rates and degree of pod-bulging were measured in the two genotypes together with assays of activities of sucrose-degrading enzymes and sugar content in pod wall and seed and evaluation of cellular pathways of phloem unloading in seed coat using a symplasmic fluorescent dye, 5(6)-carboxyfluorescein (CF).

Key Results

Activities of cell wall, cytoplasmic and vacuolar invertases (CWIN, CIN and VIN) were significantly smaller in pod walls of ‘282’ than in ‘121’ at 10 dpa onwards. Low INV activities were associated with weak pod wall growth of ‘282’. In seed coats, CF was confined within the vasculature in ‘282’ but moved beyond the vasculature in ‘121’, indicating apoplasmic and symplasmic phloem unloading, respectively. Higher CWIN activity in ‘282’ seed coats at 6–8 dpa correlated with high hexose concentration in embryos and enhanced early seed growth. However, CWIN activity in ‘282’ decreased significantly compared with ‘121’ from 10 dpa onwards, coinciding with earlier commencement of nuclei endoreduplication in their embryos.

Conclusions

The study shows genotypic differences between ‘bulging pod’ and ‘non-bulging’ phenotypes of asparagus bean in sucrose metabolism in relation to the pathway of phloem unloading in developing seed coats, and to pod and seed growth. Low INV activity in pod wall corresponds to its shortened and weak growth period; by contrast, the apoplasmic path in the seed coat is associated with high CWIN activity and strong early seed growth.