Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family. A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M_r of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patterns are discussed with respect to the probable function of the gene products. Received: 27 March 1997 / Accepted: 20 May 1997     

Expression of a cauliflower tonoplast aquaporin tagged with GFP in tobacco suspension cells correlates with an increase in cell size

In plants, vacuoles are essential organelles that undergo dynamic volume changes during cell growth due to rapid and high flow of water through tonoplast water-carrying channels composed of integral proteins (tonoplast aquaporins). The tonoplast BobTIP26-1 from cauliflower has previously been shown to be an efficient active aquaporin in Xenopus leavis oocytes. In this study we used tobacco (Nicotiana tabacum cv. Wisconsin 38) suspension cells to examine the effect of BobTIP26-1 expression. In order to follow the intracellular localisation of the protein in real time, the gfp sequence was fused downstream to the BobTIP26-1 coding region. The fusion protein BobTIP26-1::GFP is less active than BobTIP26-1 by itself when expressed in Xenopus oocytes. Nevertheless, this fusion protein is well targeted to the tonoplast of the plant suspension cell when expressed via Agrobacterium co-cultivation. A complex tonoplast labelling is shown when young vacuolated cells are observed. The expression of the fusion protein does not affect the growth rate of the cells but increases their volume. We postulate that the increase in cell volume is triggered by the fusion protein allowing vacuolar volume increase.     

New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress

Background  

The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp.     

The yeast osmosensitive mutant fps1Delta transformed by the cauliflower BobTIP1;1 aquaporin withstand a hypo-osmotic shock

Osmoregulation plays an important role in cellular responses to osmotic stress in plants and in yeast. Aquaporins contribute to osmotic adjustment by facilitating transport of water or solutes across membranes. The tonoplastic water channel BobTIP1;1 (original name BobTIP26-1) genes are upregulated during dessication stress in cauliflower meristematic tissue. To investigate the physiological importance of BobTIP1;1, we expressed it in a Saccharomyces cerevisiae osmosensitive mutant fps1Delta. We showed that the defect in the yeast glycerol plasma membrane transporter is complemented by a plant cDNA encoding the aquaporin BobTIP1;1 which is localized in the vacuolar membrane of the complemented yeast cells. To our knowledge, this is the first example of a plant aquaporin for which localization in the vacuolar membrane of yeast cells is related to an osmoresistant phenotype under hypo-osmotic shock.     

A γ-TIP cross-reacting protein is abundant in the cortex of soybean N2-fixing nodules

The distribution and abundance of tonoplast intrinsic protein (γ-TIP), a putative aquaporin which is abundant in the tonoplast of osmoregulated pulvinus motor cells, were determined in nodules of Glycine max (L.) Merr. using chemical fixation and immunolocalization. This protein was highly expressed in the tonoplast of the inner cortical cells of the nodules but poorly expressed in the vascular transfer cells and in infected cells. It is concluded that the differentiation of the inner cortical cells of the nodules like that of pulvinus motor cells, is accompanied by an increased expression of γ-TIP. This result is consistent with our previous hypothesis that a reversible exchange of intercellular water by the inner cortical cells plays a role in the regulation of nodule conductance to O₂ diffusion, and hence in subsequent N₂-fixing activity. Received: 7 February 1998 / Accepted: 22 May 1998     

Differential pH restoration after ammonia-elicited vacuolar alkalisation in rice and maize root hairs as measured by fluorescence ratio

Changes of vacuolar pH in hair cells of young rice (Oryza sativa L.) and maize (Zea mays L.) roots were measured after ammonia application at various levels of external pH. After loading the pH-sensitive, fluorescent dye Oregon green 488 carboxylic acid 6-isomer into the vacuoles of root hairs, ratiometric pH data of high statistical significance were obtained from root hair populations comprising hundreds of cells. The pH of the vacuole at external pH 5.0 was 5.32 ± 0.08 (±SD, n= 15) and 5.41 ± 0.13 (±SD, n= 15) in rice and maize, respectively. A moderate external ammonia concentration of 2 mM led to vacuolar alkalisation at both, low (pH 5.0) and high (pH 7.0–9.0) external pH, presumably due to NH₃ permeation into the vacuole. With increasing external pH, ammonia application did not cumulatively increase vacuolar pH. In rice, the increase in vacuolar pH ranged from 0.1–0.8 pH units; in maize a more constant increase of 0.5 pH units was observed. The vacuolar pH increase was efficiently depressed in rice (especially at high external pH), but not in maize. Inhibition of the tonoplast H⁺-ATPase by concanamycin A raised vacuolar pH and increased the ammonia-elicited vacuolar alkalisation in both species, proving that vacuolar H⁺-ATPase activity counters the ammonia-elicited alkalisation effect. However, even under conditions of vacuolar H⁺-ATPase inhibition, rice was still able to restore an ammonia-elicited pH increase. High vacuolar pH levels as found in maize under conditions of high NH₃ influx may derive from inefficient cytosolic ammonia assimilation and tonoplast proton pumping. Thus, in maize, prolonged reduction of the proton gradient between the cytosol and the vacuole may play an important role in NH₃ toxicity. Received: 12 September 1997 / Accepted: 19 January 1998     

Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs

Plant aquaporins occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We cloned cDNAs for plasma-membrane aquaporins (PAQ1, 1b, 1c, 2, 2b, and 2c) of radish (Raphanus sativus L.). The amino acid sequences of the PAQs showed on average 63% sequence identity. Their sequences were 23% identical to those of tonoplast aquaporins (γ- and δ-VM23). A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In other organs, the mRNA level of each isoform varied according to the organ. In petals, stamens, pistils and sepals of flowers, the levels of PAQ1, 1b, 1c and γ-VM23 mRNAs were high, and mRNAs of all aquaporins except for δ-VM23 were detected at high levels in pericarps. The protein levels of aquaporins on the basis of the membrane protein were determined by immunoblotting. Proteins PAQ1 and VM23 were detected in every organ except for the mature petiole. The PAQ2 protein level was especially high in green cotyledons and leaves, but was extremely low in seedling cotyledons and hypocotyls. Proteins PAQ1, PAQ2 and VM23 were highly accumulated in growing pericarps, but not in the immature seeds. These results indicate that the gene expression of the aquaporin isoforms was individually regulated in an organ- and tissue-specific manner, and that the amounts of aquaporin protein, especially PAQ2, are regulated in certain tissues at the translational level and by the rate of protein turnover. Received: 10 February 2000 / Accepted: 30 June 2000     

Life with and without AtTIP1;1, an Arabidopsis aquaporin preferentially localized in the apposing tonoplasts of adjacent vacuoles

The Arabidopsis thaliana Tonoplast Intrinsic Protein 1;1 (AtTIP1;1) is a member of the tonoplast aquaporin family. The tissue-specific expression pattern and intracellular localization of AtTIP1;1 were characterized using GUS and GFP fusion genes. Results indicate that AtTIP1;1 is expressed in almost all cell types with the notable exception of meristematic cells. The highest level of AtTIP1;1 expression was detected in vessel-flanking cells in vascular bundles. AtTIP1;1-GFP fusion protein labelled the tonoplast of the central vacuole and other smaller peripheral vacuoles. The fusion protein was not found evenly distributed along the tonoplast continuum but concentrated in contact zones of tonoplasts from adjacent vacuoles and in invaginations of the central vacuole. Such invaginations may result from partially engulfed small vacuoles. A knockout mutant was isolated and characterized to gain insight into AtTIP1;1 function. No phenotypic alteration was found under optimal growth conditions indicating that AtTIP1;1 function is not essential to the plant and that some members of the TIP family may act redundantly to facilitate water flow across the tonoplast. However, a conditional root phenotype was observed when mutant plants were grown on a glycerol-containing medium.     

Control of Volume and Turgor in Stomatal Guard Cells

Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca²⁺-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K⁺(Rb⁺) in response to the “drought” hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl₂ or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.     

Cortical cell death, cell proliferation, macromolecular movements and rTip1 expression pattern in roots of rice (Oryza sativa L.) under NaCl stress

The mode of action of NaCl in terms of cell proliferation and cell death was examined in seminal roots of rice plants (Oryza sativa L.). Salt/sodium chloride was inhibitory to cell number increase and to cell death in cortical tissue, whereas final cortical cell size was the same as in control roots that were not exposed to NaCl. It seems that NaCl may stimulate the transition phase from cell division to cell elongation. Further analysis of the role of NaCl in the suppression of cortical cell death was confined to a delay in the early stage of cell collapse, which was caused by tonoplast disruption, and plasma-membrane destruction. Sodium chloride did not have any effect on the cell-to-cell movement of macromolecules in the root cortex. In-situ hybridization studies indicated that expression of the gene for tonoplast intrinsic protein (rTip1) was localized predominantly in the epidermal and exodermal cells as well as in metaxylem cells in seminal roots. Upon NaCl treatment, the intensity of rTip1 gene expression was raised in the cortical parenchyma, suggesting that salt plays a role in the rapid onset of cell elongation. Received: 2 April 1998 / Accepted: 18 September 1998     

Increased Expression of Vacuolar Aquaporin and H+-ATPase Related to Motor Cell Function in Mimosa pudica L

Mature motor cells of Mimosa pudica that exhibit large and rapid turgor variations in response to external stimuli are characterized by two distinct types of vacuoles, one containing large amounts of tannins (tannin vacuole) and one without tannins (colloidal or aqueous vacuole). In these highly specialized cells we measured the abundance of two tonoplast proteins, a putative water-channel protein (aquaporin belonging to the [gamma]-TIPs [tonoplast intrinsic proteins]) and the catalytic A-subunit of H+-ATPase, using either high-pressure freezing or chemical fixation and immunolocalization. [gamma]-TIP aquaporin was detected almost exclusively in the tonoplast of the colloidal vacuole, and the H+-ATPase was also mainly localized in the membrane of the same vacuole. Cortex cells of young pulvini cannot change shape rapidly. Development of the pulvinus into a motor organ was accompanied by a more than 3-fold increase per length unit of membrane in the abundance of both aquaporin and H+-ATPase cross-reacting protein. These results indicate that facilitated water fluxes across the vacuolar membrane and energization of the vacuole play a central role in these motor cells.     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The cytological sequence of senescence-related changes in coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) was studied using fluorescence and electron microscopy. The coleoptiles reach full size 3 d after sowing, then rapidly senesce and wither completely by day 7. The interveinal region in cross-sections taken 1 mm from the tip of the coleoptile was selected for this analysis. Fluorescence microscopy using samples embedded in Technovit 7100 resin, electron microscopy and immunoelectron microscopy using DNA-specific antibodies were used to elucidate the sequence of senescence-related events. These occur in the following order: (i) degradation of the chloroplast DNA (cpDNA); (ii) condensation of the nucleus in conjunction with a decrease in the size of the dense-chromatin region, shrinkage of the chloroplast, degradation of ribulose-1, 5-bisphosphate carboxylase/oxygenase, dilation of the thylakoid membranes, increase in size and number of osmiophilic globules, condensation of the cytoplasm; (iii) disorganization of the nucleus, degeneration of the tonoplast; (iv) complete loss of the cytoplasmic components, distortion of the cell wall, invasion of microorganisms into the intercellular spaces and ultimately into the cell itself. The mitochondria maintain their ultrastructural integrity and a constant level of mitochondrial DNA throughout senescence. In young mesophyll cells, invagination of the tonoplast into the vacuole frequently occurs. This occasionally includes cytoplasmic material, which is digested in the vacuole as senescence proceeds. Immunoelectron microscopy suggests that cpDNA degradation involves rough digestion first, rather than rapid, direct decomposition of the DNA into nucleotides. The fragmented cpDNA is then dispersed throughout the chloroplast and cytoplasm. Received: 9 April 1998 / Accepted: 11 June 1998     

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH

In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n = 3) and cut into slices (mean slice weight = 13.85 ± 0.20 mg; mean slice thickness = 515.00 ± 2.33 μm) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50, and 500 ng/ml for 6 h at 32°C. Viability of the tissue was maintained based on histological integrity and lack of appreciable lactate dehydrogenase in the medium. The production and release of testosterone (T) and estradiol-17β (E2) into the medium was measured following in vitro culture. The addition of oLH increased T and E2 at least 400% and 120%, respectively, over the 0-ng oLH control cultures. Testicular gene expression was assessed with in situ hybridization methodology for steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs. In situ hybridization revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA in Leydig cells. No differences were observed for the expression of PDE3B or ODF2 genes in seminiferous tubules among treatment groups as expected. These results demonstrate the value of in vitro culture of the precision-cut tissue slices for studies of the regulation of steroidogenesis and gene expression in the stallion testes.     

Calcium changes and the response to methyl jasmonate in rice lodicules during anthesis

Summary. Potassium pyroantimonate precipitation was used to locate loosely bound calcium in rice (Oryza sativa L.) lodicules before and after anthesis, and flowering of panicles was accelerated by treatment with methyl jasmonate. From 1 day to 4 h before anthesis, the number of calcium precipitates in the cell walls and vacuole membranes decreased gradually, whereas they increased remarkably in the cytoplasm and nucleolus. At the beginning of anthesis, the number of calcium granules in lodicules reduced sharply, but there was a large accumulation of flocculent precipitates in the vacuoles. After anthesis, the flocculent precipitates decreased in number until they disappeared, whereas the granular precipitates started to accumulate once again. The rice florets treated with 2 mM methyl jasmonate were induced to open within 10–30 min and they then closed 0.5–1 h later. The nucleolus, cytoplasm, and vacuole membrane of the lodicule cells contained many calcium granules during flowering, although the cell walls lacked calcium. At 1 h after treatment, the number of calcium granules had decreased, while flocculent precipitates were regularly observed in the nondegenerated cells. At 6 h after treatment, calcium grains started to reappear in the cell walls. These changes in calcium precipitates before and after anthesis indicate that the opening and closing of florets correlates with the calcium level in lodicule cells. In addition, excised panicles, with florets judged to be nearing anthesis, were soaked in 2–200 mM EGTA solution for 2 min after treatment with 2 mM methyl jasmonate. The results indicate that EGTA had an antagonistic effect on the methyl jasmonate-induced floret opening in rice. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Association of nickel versus transport of cadmium and calcium in tonoplast vesicles of oat roots

The plant vacuole has long been suspected of being a site for accumulation of Ni in plant roots, but testing this hypothesis directly by vacuole isolation is technically difficult and has not been reported. Here, we have attempted to determine if Ni can be transported into isolated oat (Avena sativa L.) root tonoplast vesicles as an alternative approach toward understanding the importance of the vacuole in Ni accumulation in roots. We found that, in contrast to Ca and Cd, Ni did not affect the proton gradient of vesicles (MgATP energized or artificially created), and further, that Cd/H antiport activity was not affected by the presence of Ni. Nickel was associated with vesicles, but relative rates of accumulation/association of metals with vesicles were Ca > Cd Ni. Protonophores and the potential Ni ligands citrate and histidine, and nucleoside triphosphates or PPi did not stimulate Ni association with vesicles. Comparison of Ni versus Ca and Cd associated with vesicles using various membrane perturbants indicated that while Ca and Cd are rapidly and principally antiported to the vesicle sap, Ni is only slowly associated with the membrane in a not-easily dissociated condition. Our results indicate the absence of an Ni/H antiport or Ni-nucleotide-dependent pump in oat root tonoplasts, and support the contention that the vacuole is not a major compartment for Ni accumulation in oat roots. Received: 2 June 1997 / Accepted: 17 July 1997     

Altered ultrastructure of cells of sunflower leaves having low water potentials

Summary Desiccation-induced alterations in cell structure were investigated in sunflower (Helianthus annum L.) leaves using light and electron microscopy. Desiccation was imposed by withholding water from the tissue, and all tissue fixation was carried out under isosmotic conditions. In addition to shrinkage of the vacuoles and intercellular spaces caused by water loss, the significant features of cell desiccation were the appearance of lipid droplets and vesicles close to dictyosomes, and plasmalemma and/or tonoplast breakage in the mesophyll cells. Breakage was followed by massive loss of cell organelles except for the thylakoid membranes of the chloroplasts, which retained much of their integrity even in the air-dried state. Plasmalemma and tonoplast disruption began in a few cells at water potentials of — 15 bars (relative water contents of 47%) and went to completion below —26 bars (relative water contents less than 28%) in the leaf mesophyll. Typically in this tissue, net photosynthesis becomes zero and the tissue becomes increasingly incapable of full rehydration at water potentials below — 20 bars. By contrast, water potentials of — 26 bars had no detectable effects on the phloem tissue. Structural alterations were little influenced by the rapidity of desiccation (a few minutes to as long as four days). It was concluded that desiccation-induced changes in cell structure are tissue-specific and occur on a cell-by-cell basis rather than in all cells of a tissue at once. The concentration of the cytoplasm and the disruption of the plasmalemma and/or tonoplast seem to be central events in the alteration of cell ultrastructure by desiccation.This research was supported by NSF grant GB41314.     

Ascitic fluid and serum from rats with acute pancreatitis injure rat pancreatic tissues and alter the expression of heat shock protein 60

Acute pancreatitis (AP) is an inflammatory process in which cytokines and chemokines are involved. After onset, extrapancreatic stimuli can induce the expression of cytokines in pancreatic acinar cells, thereby amplifying this inflammatory loop. To further determine the role and mechanism of irritating agents in the pathogenesis of AP, rat pancreatic tissues were stimulated with ascitic fluid (APa) and serum (APs) from rats with AP or with lipopolysaccharide (LPS). In addition, the alteration of heat shock protein 60 (HSP60) expression was evaluated. Rat pancreas was removed and meticulously snipped to fragments. The snips were cultured for up to 48 h. During this period, the tissue viability as well as amylase and TNF-α levels in the supernatant and the HSP60 expression in the pancreatic tissue before and after stimulation by APa, APs, and LPS were assayed time-dependently. At different time-points during the culture, the viability and the amylase activity in the pancreatic tissue remained largely stable. After stimulation with APa, APs, or LPS for 1 h, the pancreatic tissues showed some damage, and this was followed by a sharp decrease in the viability accompanied by increased levels of amylase and TNF-α in the culture medium 2 or 4 h after stimulation (p < 0.05). In contrast, both the HSP60 mRNA and protein levels had a relatively high expression in the freshly prepared tissue fragments (0 h). As the culturing period was extended, the expression of HSP60 mRNA decreased only slightly; at the same time, the HSP60 protein levels decreased over a prolonged culture time, significantly so from 12 through 48 h (p < 0.05). After stimulation with APs, APa, or LPS, both the expression of HSP60 mRNA and protein in the tissue fragments increased slightly at 1 h and decreased significantly thereafter at 2 and 4 h (p < 0.05). APa, APs, or LPS induce injuries on isolated pancreatic tissues, accompanied by an altered HSP60 expression pattern in a time-dependent manner.     

Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes

 Recently, it has been shown that water fluxes across biological membranes occur not only through the lipid bilayer but also through specialized water-conducting proteins, the so called aquaporins. In the present study, we investigated in young and mature leaves of Brassica napus L. the expression and localization of a vacuolar aquaporin homologous to radish γ-tonoplast intrinsic protein/vacuolar-membrane integral protein of 23 kDa (TIP/VM 23). In-situ hybridization showed that these tonoplast aquaporins are highly expressed not only in developing but also in mature leaves, which export photosynthates. No substantial differences could be observed between different tissues of young and mature leaves. However, independent of the developmental stage, an immunohistochemical approach revealed that the vacuolar membrane of bundle-sheath cells contained more protein cross-reacting with antibodies raised against radish γ-TIP/VM 23 than the mesophyll cells. The lowest labeling was detected in phloem cells. We compared these results with the distribution of plasma-membrane aquaporins cross-reacting with antibodies detecting a domain conserved among members of the plasma-membrane intrinsic protein 1 (PIP1) subfamily. We observed the same picture as for the vacuolar aquaporins. Furthermore, a high density of gold particles labeling proteins of the PIP1 group could be observed in plasmalemmasomes of the vascular parenchyma. Our results indicate that γ-TIP/VM 23 and PIP1 homologous proteins show a similar expression pattern. Based on these results it is tempting to speculate that bundle-sheath cells play an important role in facilitating water fluxes between the apoplastic and symplastic compartments in close proximity to the vascular tissue. Received: 23 December 1999 / Accepted: 3 June 2000     

Selenium Regulates Gene Expression of Selenoprotein W in Chicken Gastrointestinal Tract

Selenoprotein W (SelW) is an existing form of selenium (Se). Se influences the levels of SelW in mammals. However, little is known about the pattern of SelW expression in the gastrointestinal tract tissue of bird. The present paper describes the effects of different dietary levels of Se on the SelW mRNA expression in the gastrointestinal tract tissue of chicken. The expression levels of SelW mRNA and the Se contents in the gastrointestinal tract tissues (glandular stomach, gizzard, duodenum, small intestine, and rectum) were determined on days 15, 25, 35, 45, and 55, respectively. The results showed that the Se contents and the SelW mRNA expression were significantly higher (p < 0.05) in the high-Se group, and the Se contents and SelW mRNA expression in the low-Se group were significantly lower (p < 0.05) than in the controls. The Se contents were the highest in the duodenum and the lowest in the rectum, while the SelW mRNA expression was the highest in the gizzard and the lowest in the rectum. In addition, the SelW mRNA levels in the gastrointestinal tract tissue were found to increase in a time-dependent manner with increasing feeding time. Furthermore, the expression of the SelW mRNA in the gastrointestinal tract tissues of chickens was found to correlate with the dietary Se concentrations, but not with the tissue Se contents.     

The Hypersensitive Reaction in Tobacco Leaf Tissue infiltrated with Pseudomonas pisi 4. Scanning Electron Microscope Studies on fractured Leaf Tissue

Leaves of tobacco infiltrated with Pseudomonas pisi were fractured at various times during the course of the hypersensitive reaction to expose cell surfaces within the tissue and mesophyll cell contents. Scanning electron microscopy of cross-fractured mesophyll cells did not reveal any gross change in internal structure during the reaction induction period (0—2 h), but breakdown of tonoplast and collapse of chloroplasts commenced at about 5 h, during the latent period. Death of the mesophyll cells was followed by condensation of cell contents, and pronounced stretching of cell walls, due to desiccation and shrinkage.Between 0—6 h after infiltration, bacteria were largely confined to cell junctions, frequently within droplets. With collapse of the host cells and release of cell fluid, numbers of bacteria increased considerably (many dividing cells), and there was a shift of bacterial distribution to the whole mesophyll cell surface. The progressive desiccation that occurred between 10—20 h prevented further bacterial increase, but numbers of bacteria remained stable. Death of bacteria commenced at about 15 h, and was accompanied by the formation of numerous surface protrusions, which detached and deposited over the whole mesophyll surface.