A brief note on the study of yieldin,a wall-bound protein that regulates the yield threshold of the cell wall

Yielding of plant-cell walls is defined by the mechanical properties of walls such as their extensibility (Φ for in vivo, φ for in vitro cell wall) and yield threshold (Y for in vivo, y for in vitro cell wall). A protein named yieldin, isolated from the cell wall of growing hypocotyls of Vigna unguiculata L. (cowpea), has been demonstrated to regulate the pH dependency of y in the cell wall of the glycerinated hollow cylinder of the cowpea hypocotyl. This mini-review outlines the process of the discovery of yieldin, and also refers to the progress of studies on yieldin: the molecular cloning of yieldin and its immunolocalization in the etiolated cowpea hypocotyl. Electronic Publication     

A brief note on growth physiology of plants

The concepts of physiological structure of plant axial organ and its main components are discussed. Their physiological meanings, in particular the role of the surface and the xylem proton pumps are highlighted: the former loosens the cell wall via acidification, and the latter produces the driving force for active uptake of water. Theoretical and experimental examination on the validity of the Lock-hart growth equation is reviewed. Development of a new experimental system, perfusible glycerinated hollow cylinder of cowpea hypocotyl, demonstrates the validity of the Lock-hart type mechanical equation even in such anin vitro system. The pH-dependency of both extensibility and yield threshold offer a strong support for the acid growth theory. A molecular model of cell wall extension is proposed on the basis of these results. The importance of growth regulation via control of the cell wall yield threshold is demonstrated as a very economical way, by an analogy with the performance of electron tube of triode type. Also augmentation of the classic acid growth theory is proposed on the basis of Katou's diagram and the Katou-Furumoto's model of active water uptake.     

The isolation of wall-bound proteins regulating yield threshold tension in glycerinated hollow cylinders of cowpea hypocotyl

To isolate and purify the factor regulating the yield threshold tension (y) through acidification of the cell wall, proteins were extracted from hypocotyls of Vigna unguiculata L. Their effects on the pH‐dependencies of the wall extensibility (φ) and y were examined with reconstitution experiments by incorporating them into the heat‐denatured glycerinated hollow cylinders (GHCs). The wall mechanical properties of the reconstituted GHCs were determined using stress–strain experiments. Only the proteins extracted with 1 kmol m^–3 of NaCl from the wall of elongation region restored the pH‐dependencies of φ and y once extinguished with heat‐denaturation, but proteins extracted from the other cell constituents or from the mature region of hypocotyl affect neither properties. Fractionation of the wall‐bound proteins by a hydrophobic column chromatography showed that the two different fractions affected φ or y independently. The sodium dodecyl sulphate‐polyacrylamide gel electrophoresis showed that the active fraction which restored the pH‐dependency of y still consists of two proteins of 30 and 32 kDa after purification by the sequential fractionation with cation‐exchange and gel filtration. These two proteins were named as ‘yieldin 30’ and ‘32’. Western blotting analysis using the rabbit‐antiserum against the cucumber expansin indicated that the yieldins are independent of cucumber expansin.     

Cell Wall Acidification and its Role in Auxin-Stimulated Growth

The role of cell wall acidification in auxin-stimulated growthwas examined in abraded hypocotyl segments of etiolated Cucumis.sativus seedlings. Acidification of the medium by these segmentswas strongly inhibited by a pretreatment and the continued presenceof 1?0 mol m^–3 vanadate, widely used as an inhibitor ofplasma membrane ATPase activity. Elongation of segments in pH6?5 buffer was almost completely inhibited by such a treatmentwith vanadate, and the promotion of growth by indole-3-aceticacid (IAA) seen in the absence of vanadate was completely abolished.However, both inhibited and uninhibited segments showed a pronouncedelongation in response to pH 4?0) buffer. In pH 4?0 buffer,in contrast to the results obtained at pH 6?5, IAA significantlypromoted growth in both the presence and absence of vanadate.The results indicate that IAA can promote growth in the absenceof endogenous acidification, but that an acid wall is necessaryfor wall loosening to occur. Key words: Acidification, auxin-stimulated growth, Cucumis sativus, vanadate     

The role of wall Ca2+ in the regulation of wall extensibility during the acid-induced extension of soybean hypocotyl cell walls

We examined the acid-facilitated yielding properties of cell walls of soybean hypocotyls and the effects of Ca(2+) upon the properties by stress-strain analyses using glycerinated hollow cylinders (GHCs) from the elongating regions of the hypocotyls. Stress-extension rate curves of native GHCs showed characteristic changes with pH, all indicating the existence of yield threshold tension (y) as well as wall extensibility (phi), i.e. a downward shift of y and an increase in phi with wall acidification. The acid-induced downward shift of y was inhibited by boiling of GHCs. In contrast, a considerable increase in phi with acidification remained even after boiling. This indicates that phi consists of two components, i.e. heat-sensitive and heat-resistant, both being pH sensitive. A Ca(2+) chelator (Quin 2) dramatically increased phi at a neutral pH. Subsequent addition of Ca(2+) or ruthenium red suppressed the chelator-induced increase in phi. These findings suggest that wall Ca(2+) plays an important role in the regulation of wall extensibility during the acid-induced wall extension by reacting with carboxyl groups of wall pectin.     

Inhibition of Synthesis of Peroxidases and Some Proteins by Heliangine in Phaseolus mungo Hypocotyl Sections

The peroxidase activity in Phaseolus mungo hypocotyl sectionsfloated on water decreased during the first 3 h and then increasedagain. The activity of this enzyme was reduced by heliangineand cyeloheximide in vivo, but not in vitro. Peroxidase activityis inhibited by heliangine and cycloheximide since the enzymeis not synthesized. Disk gel electrophoresis studies revealed that at least sixspecies of proteins, soluble in Tris-HCl, pH 7.6, were synthesizedin Phaseolus hypocotyl sections during 24 h incubation on water.Heliangine inhibited the synthesis of two of them, a speciesof peroxidase and another protein. Heliangine inhibited the incorporation of radioactive leucineinto the cell wall fraction, suggesting that it inhibits synthesisof cell wall proteins. It did not, however, inhibit the incorporationof labelled proline into the cell wall fraction. The resultssuggest that heliangine inhibits the synthesis of only someproteins.     

Two proteins regulate the cell wall extensibility and the yield threshold in glycerinated hollow cylinders of cowpea hypocotyl

Glycerinated hollow cylinders of hypocotyl segments excised from the elongation region of cowpea seedlings were heated for 15s in 50% glycerol at 70, 80 or 90°C. Their in vitro yield threshold tension (y) and extensibility (φ) were determined by stress-strain experiments under the perfusion of solutions of pH 4·0 or 6·2. The decrement in y and the increment in φ with acidification were extinguished at 80 and 90°C, respectively. Moreover, such changes in φ and y with acidification were prevented by proteinase treatment for 6 and 10 h, respectively. These results suggest that these two cell wall mechanical properties are controlled, respectively, by two functional proteins activated by acid.     

Subcellular Location of Phosphoglucomutase and UDP Glucose Pyrophosphorylase in Avena Coleoptiles

Extraction of phosphoglucomutuse and UDP glucose pyrophosphorylase from oat coleoptiles using both aqueous and non-aqueous systems showed that both enzymes are largely soluble. Phosphoglucomutase was completely absent from the cell wall fraction. The inhibition of phosphoglucomutase by peroxyacetyl nitrate was confirmed and the enzyme in the subcellular participate fractions was shown to be more inhibited than that in the soluble fraction. UDP glucose pyrophosphorylase was not inhibited by peroxyacetyl nitrate or ozone in vivo but was inhibited by peroxyacetyl nitrate in vitro. The SH reagents, iodoacetamide and p-chloromercuribenzoate, inhibited phosphoglucomutase severely but UDP glucose pyrophosphorylase moderately or not at all.     

Cytosine deaminase expressing human mesenchymal stem cells mediated tumour regression in melanoma bearing mice

Background

Previously, we validated capability of human adipose tissue‐derived mesenchymal stem cells (AT‐MSC) to serve as cellular vehicles for gene‐directed enzyme prodrug molecular chemotherapy. Yeast fusion cytosine deaminase : uracil phosphoribosyltransferase expressing AT‐MSC (CD_y‐AT‐MSC) combined with systemic 5‐fluorocytosine (5FC) significantly inhibited growth of human colon cancer xenografts. We aimed to determine the cytotoxic efficiency to other tumour cells both in vitro and in vivo.

Methods

CD_y‐AT‐MSC/5FC‐mediated proliferation inhibition against a panel of human tumour cells lines was evaluated in direct and indirect cocultures in vitro. Antitumour effect was tested on immunodeficient mouse model in vivo.

Results

Although culture expansion of CD_y‐AT‐MSC sensitized these cells to 5FC mediated suicide effect, expanded CD_y‐AT‐MSC/5FC still exhibited strong bystander cytotoxic effect towards human melanoma, glioblastoma, colon, breast and bladder carcinoma in vitro. Most efficient inhibition (91%) was observed in melanoma A375 cell line when directly cocultured with 2% of therapeutic cells CDy‐AT‐MSC/5FC. The therapeutic paradigm of the CDy‐AT‐MSC/5FC system was further evaluated on melanoma A375 xenografts on nude mice in vivo. Complete regression in 89% of tumours was achieved when 20% CD_y‐AT‐MSC/5FC were co‐injected along with tumour cells. More importantly, systemic CD_y‐AT‐MSC administration resulted in therapeutic cell homing into subcutaneous melanoma and mediated tumour growth inhibition.

Conclusions

CD_y‐AT‐MSC capability of targeting subcutaneous melanoma offers a possibility to selectively produce cytotoxic agent in situ. Our data further demonstrate beneficial biological properties of AT‐MSC as a cellular vehicle for enzyme/prodrug therapy approach to molecular chemotherapy. Copyright © 2008 John Wiley & Sons, Ltd.     

Wall yield threshold and effective turgor in growing bean leaves

The rate of cell enlargement depends on cell-wall extensibility (m) and on the amount of turgor pressure (P) which exceeds the wall yield threshold (Y). The difference (P-Y) is the growth-effective turgor (P _e). Values of P, Y and P _ehave been measured in growing bean (Phaseolus vulgaris L.) leaves with an isopiestic psychrometer, using the stress-relaxation method to derive Y. When rapid leaf growth is initiated by light, P, Y and P _eall decrease. Thereafter, while the growth rate declines in maturing leaves, Y continues to decrease and P _eactually increases. These data confirm earlier results indicating that the changes in light-stimulated leaf growth rate are primarily controlled by changes in m, and not by changes in P _e. Seedlings incubated at 100% relative humidity have increased P, but this treatment does not increase growth rate. In some cases Y changes in parallel with P, so that P _eremains unchanged. These data point out the importance of determining P _e, rather than just P, when relating cell turgor to the growth rate.Abbreviations and symbols FC fusicoccin - m wall extensibility - P turgor pressure - P _e effective turgor - RH relative humidity - Y yield threshold - _w water potential - _s osmotic potential     

2,3-Butanedione monoxime increases sensitivity to Nikkomycin Z in the budding yeast Saccharomyces cerevisiae

Summary Nikkomycin Z (NZ) is a competitive inhibitor of chitin synthase III in the yeast Saccharomyces cerevisiae. Myosin type II-deficient yeast strains (myo1) display a dramatic reduction in growth when chitin synthase III activity is inhibited by NZ, supporting the contention that actomyosin motility plays an important role in maintaining cell wall integrity. A proposed inhibitor of cortical actin polymerization in vitro, 2,3-butanedione monoxime (BDM), also inhibits growth of wild-type yeast strains at a concentration of 20 mM. In this study, we assayed for potential in vivo interplay between BDM-sensitive cell functions and cell wall chitin synthesis by testing for increased sensitivity to NZ during co-treatment with BDM at sub-inhibitory concentrations. Our results show that BDM can increase the sensitivity of yeast cells to Nikkomycin Z.     

In vivo and in vitro investigation of salt tolerance in three anthocyaninless mutants in tomato (Lycopersicon esculentum Mill.)

Growth responses of a tomato cultivar Ailsa Craig and the ah-, aw- and bls-isogenic/near isogenic lines (IL/NIL) from it were evaluated and compared at cotyledons stage under salt treatment in vivo and in vitro experiments. No differences in hypocotyl and root growth responses were detected between the anthocyanin-containing and the anthocyaninless lines within the in vivo experiments. The anthocyaninless mutants, (except in some cases the bls mutant), exhibited higher callogenic and shoot-forming capacity on both, control and salinized media. It was concluded that for this reason it would be difficult to determine the relationship between the in vivo and in vitro responses of the lines studied and as well as to evaluate the usefulness of the in vitro method in testing these lines for salt tolerance.     

Effects of membrane acting-drugs on plasmodium species and sickle cell erythrocytes

The effects of several membrane-acting drugs on malaria and sickle cell anemia was studied. In the initial experiments, propranolol and W-7 were shown to increase red cell density.In vitro, these drugs inhibited the growth ofP. falciparum. However,in vivo experiments using the murine malarial parasite,P. vinckei, demonstrated little, if any, anti-parasite activity with the doses of drugs employed. Subsequently, prostaglandin oligomeric derivatives were found to inhibit the growth ofP. falciparum in vitro andP. vinckei in vivo. Since prostaglandin oligomers inhibited the formation of dense, dehydrated cells (irreversible sickle cells), they may also have therapeutic efficacy in sickle cell anemia.     

Inhibition of the growth of human hepatoma cell line bothin vitro andin vivo by transducing CKI genep21 WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous genepCEP-p21 ^WAF-1 into human hepatocellular carcinoma cell bothin vitro andin vivo. Afterin vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. Thein vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ±0.043) g, while that of the control group was (0.281 ±0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous genepCEP-p21 ^WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy bothin vivo andin vitro.     

Role of K+ in leaf growth: K+ uptake is required for light-stimulated H+ efflux but not solute accumulation

The stimulation of dicotyledonous leaf growth by light depends on increased H⁺ efflux, to acidify and loosen the cell walls, and is enhanced by K⁺ uptake. The role of K⁺ is generally considered to be osmotic for turgor maintenance. In coleoptiles, auxin‐induced cell elongation and wall acidification depend on K⁺ uptake through tetraethylammonium (TEA)‐sensitive channels (Claussen et al., Planta 201, 227–234, 1997), and auxin stimulates the expression of inward‐rectifying K⁺ channels ( Philippar et al. 1999) . The role of K⁺ in growing, leaf mesophyll cells has been investigated in the present study by measuring the consequences of blocking K⁺ uptake on several growth‐related processes, including solute accumulation, apoplast acidification, and membrane polarization. The results show that light‐stimulated growth and wall acidification of young tobacco leaves is dependent on K⁺ uptake. Light‐stimulated growth is enhanced three‐fold over dark levels with increasing external K⁺, and this effect is blocked by the K⁺ channel blockers, TEA, Ba⁺⁺ and Cs⁺. Incubation in 10 mm TEA reduced light‐stimulated growth and K⁺ uptake by 85%, and completely inhibited light‐stimulated wall acidification and membrane polarization. Although K⁺ uptake is significantly reduced in the presence of TEA, solute accumulation is increased. We suggest that the primary role of K⁺ in light‐stimulated leaf growth is to provide electrical counterbalance to H⁺ efflux, rather than to contribute to solute accumulation and turgor maintenance.     

In vitro and in vivo antifungal activates of the essential oils of various plants against strawberry grey mould disease agent Botrytis cinerea

In order to investigate the effects of antifungal essential oils on postharvest decay and some quality factors of strawberry fruit, experiments were conducted under in vitro and in vivo conditions. The antifungal activates of essential oils obtained from fennel, anis, peppermint and cinnamon at concentrations 0, 200, 400, 600 and 800 μL L^?1 were investigated against Botrytis cinerea with four replications. In vitro results showed that the growth of B. cinerea was completely inhibited by fennel, cinnamon and anis essential oils at relatively low concentrations (400–800 μL L^?1). In vivo results showed that all the used essential oils at all applied concentrations caused an increase in the shelf life and inhibited of B. cinerea growth on strawberry fruits completely in comparison to the controls. The results of this study confirmed the antifungal effect of four essential oils in both in vitro and on fruit postharvest.     

Tomato-Fusarium oxysporum f. sp. lycopersici Interaction: "in vitro" Analysis of Several Possible Pathogenic Factors

The behaviour of Tomato cultures from known resistant and susceptible cultivars and lines was examined for callus growth on culture media containing increasing concentrations of Fusarium oxysporum f. sp. lycopersici race 1 culture filtrate and phytoalexin synthesis elicited by Fusarium cell wall components. A strict correlation was found between in vivo resistance to the fungus and in vitro hypersensitive response and phytoalexin induction. On the other hand in vitro tolerance to toxic filtrate does not seem in this case a good indicator of in vivo resistance to the pathogen.     

Impaired growth in transgenic plants over-expressing an expansin isoform

Expansins are cell wall proteins characterised by their ability to stimulate wall loosening during cell expansion. The expression of some expansin isoforms is clearly correlated with growth and the external application of expansins can stimulate cell expansion in vivo in several systems. We report here the expression of a heterologous expansin coding sequence in transgenic tomato plants (Lycopersicon esculentum Mill.) under the control of a constitutive promoter. In some transgenic lines with high levels of expansin activity extractable from cell walls, we observed alterations of growth: mature plants were stunted, with shorter leaves and internodes, and dark-grown seedlings had shorter and wider hypocotyls than their wild-type counterparts. Examination of hypocotyl sections revealed similar differences at the cellular level: cortical and epidermal cells were shorter and wider than those from wild-type seedlings. The observed stimulation of radial expansion did not compensate for the decreased elongation, and overall growth was reduced in the transgenics. As this observation can seem paradoxical given the known effect of expansins on isolated cell walls, we examined the mechanical behaviour of transgenic tissue. We measured a decrease in hypocotyl elongation in response to acidic pH in the transformants. This result may account for the alterations in cell expansion, and could itself be explained by a reduced susceptibility of transgenic cell walls to expansin action.     

Influence of pH and initial lactate concentration on the growth of Lactobacillus plantarum

Summary Fermentation yields of Lactobacillus plantarum were measured at controlled pH between 4.0 and 8.0 and initial lactate concentrations of 0–90 g/l. Optimal growth conditions at pH 6.0 without addition of lactate gave a growth rate of 0.57 h^–1 and 20 g dry biomass/mol ATP formed (Y _ATP). The pH variations resulted in a decrease in growth rate but the effect on Y _ATPwas insignificant. The addition of lactate to the medium at 0 h resulted in linear decrease in the growth rate of the culture, and all the metabolic activities were completely inhibited at 110 g/l. The Y _ATPand biomass/ substrate yield (Y _X/S) remained fairly steady up to 33 g lactate/l, beyond which both yields decreased considerably. Offsprint requests to: M. Raimbault