Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well‐developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high‐value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar‐targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N‐terminal 78‐bp‐long putative vacuolar‐targeting sequence from the N‐terminal domain of unknown function (DUF) in Triticum aestivum 6‐SFT (sucrose: fructan 6‐fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar‐targeting determinants at the N‐terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His‐tagged β‐glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar‐targeting determinant could localize recombinant proteins (r‐proteins) to the vacuole in high concentrations and such targeted r‐proteins can be purified from the juice with a few simple steps.     

Protein storage vacuole acidification as a control of storage protein mobilization in soybeans

Soybean protease C1 (EC 3.4.21.25), the subtilisin-like serine protease that initiates the proteolysis of seed storage proteins in germinating soybean [Glycine max (L.) Merrill], was localized to the protein storage vacuoles of parenchyma cells in the cotyledons by immunoelectron microscopy. This was demonstrated not only in germination and early seedling growth as expected, but also in two stages of protein storage vacuole development during seed maturation. Thus, the plant places the proteolytic enzyme in the same compartment as the storage proteins, but is still able to accumulate those protein reserves. Since soybean protease C1 activity requires acidic conditions for activity, the hypothesis that the pH condition in the protein storage vacuole would support protease C1 activity in germination, but not in seed maturation, was tested. As hypothesized, acridine orange accumulation in the protein storage vacuole of storage parenchyma cells was detected by fluorescence confocal microscopy in seedlings before the onset of mobilization of reserve proteins as noted by SDS-PAGE. Accumulation of the dye was reversed by inclusion of the weak base methylamine to dissipate the pH gradient across the vacuolar membrane. Also as hypothesized, acridine orange did not accumulate in the protein storage vacuole of those parenchyma cells during seed maturation. These results were obtained using cells separated by pectolyase treatment and also using cotyledon slices.     

Alkalinity of Neutrophil Phagocytic Vacuoles Is Modulated by HVCN1 and Has Consequences for Myeloperoxidase Activity

The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1^-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.     

Compartment acidification is required for efficient sorting of proteins to the vacuole in Saccharomyces cerevisiae.

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles. These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles. The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing. Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH. A similar type of missorting defect can be induced in wild-type cells at pH 7.5. These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.     

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.     

Vacuolar respiration of nitrate coupled to energy conservation in filamentous Beggiatoaceae

We show that the nitrate storing vacuole of the sulfide‐oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellular energy conservation. Immunostaining by antibodies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gradient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These findings show consistently that nitrate respiration contributes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation.     

Vacuole proteins in parenchyma cells of secondary phloem and xylem of Dalbergia odorifera

Summary Light- and electron-microscopic observations were made on the stem parenchyma cells of Dalbergia odorifera T. Chen (Papilionaceae), a tropical deciduous tree. In the secondary phloem of branchlet and trunk, all of the parenchyma cells except companion cells contain vacuole proteins. Only the outer secondary xylem of branchlets, but not trunk secondary xylem, has proteins in the ray parenchyma and the vasicentric parenchyma. The xylem vacuole proteins begin to accumulate at the end of the growing period and they disappear after the first flush of growth in spring. The vacuole proteins in phloem cells, particularly in the cells near the cambium, also show seasonal fluctuations. Under the electron microscope, the vacuole proteins appear as fibrous materials in aggregation or in more or less even dispersion, and they occur in the large central vacuoles during both the growth and dormant periods. According to the published studies, the stem storage proteins in the temperate trees appear as small protein-storage vacuoles or protein bodies, and the proteins in the tropical trees occur in large central vacuoles. This distinction is assumed to be related to the differences in the nature of dormancy between temperate and tropical trees.     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating Arabidopsis thaliana seeds

Plant cell vacuoles are diverse and dynamic structures. In particular, during seed germination, the protein storage vacuoles are rapidly replaced by a central lytic vacuole enabling rapid elongation of embryo cells. In this study, we investigate the dynamic remodeling of vacuolar compartments during Arabidopsis seed germination using immunocytochemistry with antibodies against tonoplast intrinsic protein (TIP) isoforms as well as proteins involved in nutrient mobilization and vacuolar acidification. Our results confirm the existence of a lytic compartment embedded in the protein storage vacuole of dry seeds, decorated by γ-TIP, the vacuolar proton pumping pyrophosphatase (V-PPase) and the metal transporter NRAMP4. They further indicate that this compartment disappears after stratification. It is then replaced by a newly formed lytic compartment, labeled by γ-TIP and V-PPase but not AtNRAMP4, which occupies a larger volume as germination progresses. Altogether, our results indicate the successive occurrence of two different lytic compartments in the protein storage vacuoles of germinating Arabidopsis cells. We propose that the first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells.     

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.     

Tonoplast intrinsic protein isoforms as markers for vacuolar functions

Plant cell vacuoles may have storage or lytic functions, but biochemical markers specific for the tonoplasts of functionally distinct vacuoles are poorly defined. Here, we use antipeptide antibodies specific for the tonoplast intrinsic proteins alpha-TIP, gamma-TIP, and delta-TIP in confocal immunofluorescence experiments to test the hypothesis that different TIP isoforms may define different vacuole functions. Organelles labeled with these antibodies were also labeled with antipyrophosphatase antibodies, demonstrating that regardless of their size, they had the expected characteristics of vacuoles. Our results demonstrate that the storage vacuole tonoplast contains delta-TIP, protein storage vacuoles containing seed-type storage proteins are marked by alpha- and delta- or alpha- and delta- plus gamma-TIP, whereas vacuoles storing vegetative storage proteins and pigments are marked by delta-TIP alone or delta- plus gamma-TIP. In contrast, those marked by gamma-TIP alone have characteristics of lytic vacuoles, and results from other researchers indicate that alpha-TIP alone is a marker for autophagic vacuoles. In root tips, relatively undifferentiated cells that contain vacuoles labeled separately for each of the three TIPs have been identified. These results argue that plant cells have the ability to generate and maintain three separate vacuole organelles, with each being marked by a different TIP, and that the functional diversity of the vacuolar system may be generated from different combinations of the three basic types.     

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.     

Heterologous expression of vacuolar H+-PPase enhances the electrochemical gradient across the vacuolar membrane and improves tobacco cell salt tolerance

Summary. The vacuolar H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) uses pyrophosphate as substrate to generate the proton electrochemical gradient across the vacuolar membrane to acidify vacuoles in plant cells. The heterologous expression of H⁺-PPase genes (TsVP from Thellungiella halophila and AVP1 from Arabidopsis thaliana) improved the salt tolerance of tobacco plants. Under salt stress, the transgenic seedlings showed much better growth and greater fresh weight than wild-type plants, and their protoplasts had a normal appearance and greater vigor. The cytoplasmic and vacuolar pH in transgenic and wild-type cells were measured with a pH-sensitive fluorescence indicator. The results showed that heterologous expression of H⁺-PPase produced an enhanced proton electrochemical gradient across the vacuolar membrane, which accelerated the sequestration of sodium ions into the vacuole. More Na⁺ accumulated in the vacuoles of transgenic cells under salt (NaCl) stress, revealed by staining with the fluorescent indicator Sodium Green. It was concluded that the tonoplast-resident H⁺-PPase plays important roles in the maintenance of the proton gradient across the vacuolar membrane and the compartmentation of Na⁺ within vacuoles, and heterologous expression of this protein enhanced the electrochemical gradient across the vacuolar membrane, thereby improving the salt tolerance of tobacco cells. Correspondence: J.-R. Zhang, School of Life Science, Shandong University, 27 Shanda South Road, Jinan, People’s Republic of China 250100.     

Vacuolar citrate/H+ symporter of citrus juice cells

We have isolated a cDNA, designated Citrus sinensis citrate transporter 1 CsCit1 encoding a novel vacuolar citrate/symporter. Immunoblots using antibodies raised against CsCit1 showed that the protein is localized to the juice sac cell vacuoles. The highest expression of CsCit1 and the amount of protein in the juice sac cell vacuoles coincided with the developmental stage at which the vacuolar citrate content began declining with the concomitant increase in vacuolar pH. Vacuoles from Sacharomyces cereviseae expressing CsCit1 displayed a citrate-dependent H⁺ efflux, and our results clearly demonstrate that CsCit1 is able to mediate the electroneutral co-transport of H⁺ and citrate ions, since the citrate-dependent H⁺ fluxes are not affected by changing the electrical potential difference across the tonoplast. The roles of CsCit1 in mediating citrate efflux from the vacuole and on citric acid homoestasis in Citrus juice sac cells are discussed. T. Shimada and R. Nakano contributed equally to this work.     

The cargo in vacuolar storage protein transport vesicles is stratified

Developing pea seeds contain two functionally distinct vacuoles--lytic vacuoles and protein storage vacuoles (PSV). The Golgi apparatus of these cells has to discriminate between proteins destined for these vacuolar compartments. Whereas it is known that sorting into the lytic vacuole is performed via the conserved clathrin-coated vesicle pathway, sorting of proteins into the protein storage vacuole remains enigmatic. In developing pea cotyledons, the major storage proteins are sorted via 'dense vesicles'. In this report we examined the sorting of a minor protein of the protein storage vacuole, the sucrose-binding-protein homolog (SBP), along the secretory pathway employing immunoelectron microscopy on cryosectioned pea cotyledons. SBP follows the same vesicular route into the PSV as the main storage proteins legumin and vicilin, via the dense-vesicles. Furthermore, legumin and SBP are sorted together into the same dense vesicle population at the stack. Although soluble cargo proteins of the dense vesicles, they show a stratified distribution in the lumen of the dense vesicles. Whereas the legumin label is equally distributed across the lumen, the SBP label is concentrated at the membrane of the vesicle. This observation is discussed with respect to a putative receptor-mediated sorting of the proteins into the dense vesicles.     

Vacuolar transporters and their essential role in plant metabolism

Following the unequivocal demonstration that plants contain at least two types of vacuoles, scientists studying this organelle have realized that the plant 'vacuome' is far more complex than they expected. Some fully developed cells contain at least two large vacuoles, with different functions. Remarkably, even a single vacuole may be subdivided and fulfil several functions, which are supported in part by the vacuolar membrane transport systems. Recent studies, including proteomic analyses for several plant species, have revealed the tonoplast transporters and their involvement in the nitrogen storage, salinity tolerance, heavy metal homeostasis, calcium signalling, guard cell movements, and the cellular pH homeostasis. It is clear that vacuolar transporters are an integrated part of a complex cellular network that enables a plant to react properly to changing environmental conditions, to save nutrients and energy in times of plenty, and to maintain optimal metabolic conditions in the cytosol. An overview is given of the main features of the transporters present in the tonoplast of plant cells in terms of their function, regulation, and relationships with the microheterogeneity of the vacuome.     

Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.     

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl₂ to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by ³¹P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na⁺ and Mg²⁺ are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K⁺ is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg²⁺ and Na⁺, show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K⁺, the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.Abbreviation NMR nuclear magnetic resonance The very cooperative help by Dr. J. Reiner with the ³¹P-NMR measurements and the technical assistance by D. Keis are gratefully acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.     

Monoclonal antibodies to barley aleurain and homologs from other plants

Barley aleurain is contained within a specific type of vacuole characterized by acidic pH and the presence of other hydrolytic enzymes. The aleurain-containing vacuole is distinct from protein storage vacuoles, and anti-aleurain antibodies serve as markers for this organelle in barley cells. Aleurain is a unique type of cysteine protease, and other plant species have genes for homologs whose sequences are highly conserved, but little is known about these homologs at the protein level. Seven monoclonal antibodies to barley aleurain were isolated, which bind to and define aleurain homologues in Arabidopsis, Petunia , and tobacco cell extracts. Interestingly, in addition to 29–32 kDa aleurain homologs, Petunia extracts contain a protein of ∼50 kDa and tobacco extracts a protein of ∼40 kDa that are recognized by multiple different mono-clonal antibodies, indicating an unexpected diversity to the aleurain protein family. Among the group of antibodies are some that efficiently immunoprecipitate metabolically labeled aleurain from barley cell extracts, and some that efficiently label aleurain in immunofluorescence assays using root tip cells. These antibodies should be useful for plant cell biologists who study vacuole biogenesis and function and sorting of proteins to specific vacuolar compartments, in barley as well as other plant species.