Ion-Exchange Properties of Cell Walls Isolated from Lupine Roots

Acid–base properties of cell walls isolated from various root tissues of 7-day-old lupine seedlings and 14-day-old lupine plants grown in various media were studied. The ion-exchange capacity of root cell walls was estimated at various pH values (from 2 to 12) and constant ionic strength (10 mM). The parameters determining the qualitative and quantitative composition of cell wall ionogenic groups along the root length and in its radial direction were estimated using Gregor's model. This model fits the experimental data reasonably well. Four types of ionogenic groups were found in the cell walls: an amino group (pK _a 3), two types of carboxylic groups (pK _a 5 and 7.3, the first being the carboxylic group of galacturonic acid), and a phenolic group (pK _a 10). The number of functional groups of each type was estimated, and the corresponding ionization constant values were calculated. It is shown that the chemical composition of the ionogenic groups was constant along the root length as well as in its radial direction and did not depend on either physiological state or root nutrition, while the number of different groups varied. The content of carboxylic groups of -D-polygalacturonic acid in the root cell walls of 14-day-old plants was shown to depend on the distance from the root tip, being maximal in the zone of lateral roots. The number of these groups was 10- and 2-fold less in the central cylinder compared to that of cortex for 14-day-old plants and 7-day-old seedlings, respectively.     

Molecular Cloning and Characterization of a Vacuolar H+-pyrophos-phatase Gene, SsVP, from the Halophyte Suaeda salsa and its Overexpression Increases Salt and Drought Tolerance of Arabidopsis

The chenopodiaceae Suaeda salsa L. is a leaf succulent euhalophyte. Shoots of the S. salsa are larger and more succulent when grown in highly saline environments. This increased growth and water uptake has been correlated with a large and specific cellular accumulation of sodium. S. salsa does not have salt glands or salt bladders on its leaves. Thus, this plant must compartmentalize the toxic Na⁺ in the vacuoles. The ability to compartmentalize sodium may result from a stimulation of the proton pumps that provide the driving force for increased sodium transport into the vacuole. In this work, we isolated the cDNA of the vacuolar membrane proton-translocating inorganic pyrophosphatase (H⁺-PPase) from S. salsa. The SsVP cDNA contains an uninterrupted open reading frame of 2292 bp, coding for a polypeptide of 764 amino acids. Northern blotting analysis showed that SsVP was induced in salinity treated leaves. The activities of both the V-ATPase and the V-PPase in Arabidopsis overexpressing SsVP-2 is higher markedly than in wild-type plant under 200 mM NaCl and drought stresses. The Overexpression of SsVP can increase salt and drought tolerance of transgenic Arabidopsis. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Shanli Guo, Haibo Yib: These authors contributed equally to this work     

Molecular Cloning and Different Expression of a Vacuolar Na+/H+ antiporter gene in Suaeda salsa Under Salt Stress

A Na⁺/H⁺ antiporter catalyzes the transport of Na⁺ and H⁺ across the tonoplast membrane. We isolated a vacuolar Na⁺/H⁺ antiporter cDNA (SsNHX1) clone from a euhalophyte, Suaeda salsa. The nuclear sequence contains 2262 bp with an open reading frame of 1665 bp. The deduced amino acid sequence is similar to that of AtNHX1 and OsNHX1 in rice, with the highest similarities within the predicted transmembrane segments and an amiloride-binding domain. Northern blot analysis shows that the expression of the S. salsa gene was increased by salt stress. The results suggest that the SsNHX1 product is likely a Na⁺/H⁺ antiporter and may play important roles in the salt tolerance of S. salsa. This revised version was published online in July 2006 with corrections to the Cover Date.     

X-ray microanalysis of ion distribution within root cortical cells of the halophyte Suaeda maritima (L.) Dum.

The ion content of compartments within cortical cells of mature roots of the halophyte Suaeda maritima grown at 200 mol·m^-3 NaCl has been studied by X-ray microanalysis of freeze-substituted thin sections. Sodium and Cl were found in the vacuoles at about four-times the concentration in the cytoplasm or cell walls, whereas K was more concentrated in the cell walls and cytoplasm than in vacuoles. The vacuolar Na concentration was 12- to 13-times higher than that of K. The Na concentration of cell walls of cortical cells was about 95 mol·m^-3 of analysed volume. The cytoplasmic K concentration within the mature cortical cells was estimated to be 55 mol·m^-3 of analysed volume.     

Specific regulation of SOD isoforms by NaCl and osmotic stress in leaves of the C3 halophyte Suaeda salsa L

The halophyte Suaeda salsa L., exposed to different NaCl concentrations (100 and 400 mmol/L) and polyethylene glycol (isoosomotic to 100 mmol/L NaCl) containing nutrient solutions under normal or K+-deficient conditions for 7 days, was used to study effects of NaCl salinity and osmotic stress on chlorophyll content, chlorophyll fluorescence characteristics, malonedialdehyde (MDA) content, and superoxide dismutase (SOD) isoform activities. Photosynthetic capacity was not decreased by NaCl treatment, indicating that S. salsa possesses an effective antioxidative response system for avoiding oxidative damage. Seven SOD activity bands were detected in S. salsa leaf extracts, including an Mn-SOD and several isoforms of Fe-SOD and CuZn-SOD. It turned out that NaCl salinity and osmotic stress lead to a differential regulation of distinct SOD isoenzymes. This differential regulation is suggested to play a major role in stress tolerance of S. salsa.     

Salt tolerance in the halophyte Suaeda maritima (L.) Dum. The influence of the salinity of the culture solution on leaf starch and phosphate content

Abstract. The starch concentration in mature leaves of the halophyte Suaeda maritima increased from 4.7 to 7.3 mg mg⁻¹ chlorophyll when sodium chloride (680 mol M⁻³) was added to the solution in which the plants were grown. This effect of salinity on the starch: chlorophyll ratio was greater in young than in old leaves. Electron micrographs showed the starch to be in the chloroplasts and this was confirmed by measurements on isolated chloroplasts. Total phosphorus concentration (mg mg⁻¹ chlorophyll) in leaves of all ages from plants of S. maritima decreased on salinization of the growth medium suggesting an inverse relationship between phosphorus and starch concentrations. However, although leaf starch concentration varied with leaf age, phosphorus concentration did not. The cause of starch accumulation in chloroplasts at salinities which are optimal for growth (340 mol m⁻³) remains unclear.     

Responses of some genetically diverse lines of chick pea (Cicer arietinum L.) to salt

The salt tolerance of three tolerant accessions of chick pea, CM 663, 10130 and 10572 and three sensitive accessions 10582, 12908 and 12909 selected at the germination and seedling stage was assessed at the adult stage using sand culture salinized with 0, 40 or 80 mol m^-3 NaCl. The two tolerant accessions, CM 663 and 10572 and one sensitive, 12908 showed consistent correlation between the degrees of salt tolerance at the early growth stages and adult stage as the former two produced significantly higher seed yield compared with the other accessions and the latter did not survive till seed setting in the salt treatments. By contrast 10130 which was found relatively salt tolerant at the two early growth stages could not survive in 40 mol m^-3 NaCl till seed setting. Similarly two sensitive accessions, 10582 and 12909 not only survived at the adult stage but produced some yield as well. On the basis of performance of the six accessions at three different stages, accessions CM 663 and 10572 can be categorised as relatively salt tolerant, 12908 as sensitive and 10130, 10582 and 12909 as moderately tolerant. The tolerant accession CM 663 had high Na⁺ and Cl^- in the leaves but maintained high K:Na ratios and high K⁺ versus Na⁺ selectivity. This accession had relatively low leaf osmotic potential which may be due to its high accumulation of Na⁺ and Cl^- in the leaves. By contrast the second tolerant accession 10572 had lowest Na⁺ and moderate Cl^- in the leaves.of all accessions but had highest K⁺ versus Na⁺ selectivity, although its leaf K:Na was intermediate. It had also relatively low osmotic potential which cannot be related to different ions determined in this study. The salt sensitive accession 12908 had high leaf Na⁺ and moderate Cl^- but had very low K:Na ratio (less than one) and K⁺ versus Na⁺ selectivity. The remaining accessions as a whole did not show any consistent pattern of uptake of different ions. The positive correlation between the degree of salt tolerance at different growth stages do exist in some accessions of chick pea examined in the present study, but for others in which no positive correlation was observed suggests that a combination of certain characters can be used as selection criterion for improving salt tolerance in chick pea.     

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (S _i) and the swelling coefficient (K ^cw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pH_i = f (S _i), the Gregor's equation was employed. It was shown that the Gregor's model fits well the experimental data. The total number of the cation exchange (S _t ^cat) and the anion exchange (S _t ^an) groups were determined in the root cell walls. The number of the functional group of each type (S ^j) was estimated, and the corresponding values of pK _a ^j were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferich's equation [pK _a ^j = f (C ^K+)] were determined. It was found that the swelling of root cell walls changes with pH, C ^K+ and strongly depends on plant species. Within the experimental pH and C ^K+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (S ^j, pK _a ^j, K ^cw), the equation {pK _a ^j = f (C^K+)} and the dependencies {K ^cw = f (C^K+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.     

Molecular cloning and characterization of betaine aldehyde dehydrogenase gene from Suaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco

cDNA encoding betaine aldehyde dehydrogenase (BADH) from the halophyte Suaeda liaotungensis has been cloned, sequenced and expressed in tobacco (Nictiana tabacum 89). The full-length cDNA is 1506 base pairs (bp) long and encodes a 502 amino-acid polypeptide. The cDNA fragment coding for the mature enzyme was cloned into vector pCAMBIA-1301 for expression in tobacco. Southern blotting analysis showed that that the Badh gene was integrated into the genome of tobacco. Tobacco expressing BADH survived on MS medium containing 200 mM NaCl, whereas the untransformed plants turned yellow after about 20 d and died.     

Heterogeneity of Maize Root Mitochondria from Plants Grown in the Presence of Ammonium

Mitochondria isolated from root tissue of maize plants grown on a modified Knop solution containing 10.9 mM nitrate ± 7.2 mM ammonium were purified on the discontinuous Percoll density gradient with polyvinylpyrrolidone (PVP) added. The presence of PVP allowed separation of several mitochondrial fractions of a different density. Contrary to mitochondria isolated from plants grown in the presence of nitrate alone, revealing only two fractions, the mitochondria from NH₄ ⁺/NO₃ ^–-plants were distributed in four fractions. Total amount of mitochondria, as well as specific activities of some nitrogen metabolism enzymes and tricarboxylic acid (TCA) cycle enzymes of all mitochondrial fractions, and respiratory activities of two lower density fractions isolated from plants grown on mixed nitrogen were higher in comparison to mitochondria from nitrate-grown plants.     

Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1

Transgenic rice plants co-expressing the Suaeda salsa SsNHX1 (vacuolar membrane Na⁺/H⁺ antiporter) and Arabidopsis AVP1 (vacuolar H⁺-PPase) showed enhanced salt tolerance during 3 d of 300 mM NaCl treatment under outdoor growth conditions. These transgenic rice seedlings also grew better on MS medium containing 150 mM NaCl compared to SsNHX1-transformed lines and non-transformed controls. Measurements on isolated vacuolar membrane vesicles derived from the salt stressed SsNHX1+AVP1-transgenic plants demonstrated that the vesicles had increased V-PPase hydrolytic activity in comparison with the Ss-transgenics and non-transgenics. Moreover the V-PPase activity was closely related to the development period of the SA-transgenic seedlings and markedly higher in 3-week-old seedlings than in 5-week-old seedlings. Statistic analysis indicated that the SA-transgenic rice plants contained relatively more ions with higher K⁺/Na⁺ ratio in their shoots compared to the SsNHX1-transformed lines upon salt treatment. Furthermore, these SA-transformants also exhibited relatively higher level of photosynthesis and root proton exportation capacity whereas reduced H₂O₂ generation in the same plants. In general, these results supported the hypothesis that simultaneous expression of the SsNHX1 and AVP1 conferred greater performance to the transgenic plants than that of the single SsNHX1.Feng-Yun Zhao and Xue-Jie Zhang contributed equally to this work     

Trichomes: their Diversity,Distribution and Density in Acclimatization of Teak (Tectona grandis L.) Plants Grown in vitro

Diversity, distribution and density of trichomes were studied in relation to other morphological and physiological parameters of in vivo and in vitro teak (Tectona grandis L.) plants with the objective to understand their role in hardening and acclimatization of micropropagated plants. Less diversity and scanty distribution of trichomes of in vitro leaves in comparison to the in vivo ones were correlated with water loss of the former plants leading to low rate of survival after transplantation. The probable cause for absence of certain types of trichome in in vitro plants was also discussed.     

Swelling of Root Cell Walls as an Indicator of Their Functional State

The swelling capacity of cell walls isolated from different parts of lupine root was investigated. The water content in fragments of intact roots (Q) and swelling coefficient of standardized samples of cell walls (K^cw) were determined, and the dependences of Q and Kcw on the distance from the root tip (L) were plotted. It was shown that the change in Q value along the stretch of the lupine root reaches its maximum at distances of 1.5-6 cm or 7-12 cm from the root tip in 7-day-old and 14-day-old seedlings, respectively, whereas the K^cw value distribution over the root length is virtually invariable. In the radial direction, both the Q and K^cw values in cortex tissues are about twice higher than in the central cylinder. In our opinion, the changes of both Q and K^cw in the radial direction are associated with different degrees of cross-linking between polymer chains in cell wall structures of root cortex and central cylinder. The results of measurement of the K^cw value are consistent with the widely accepted mechanisms of water transport in roots in the radial direction. These data show that water transport through apoplast to the border between the cortex and central cylinder is accompanied by an increase in the resistance to water flow. Among other factors, this increase is due to a greater degree of cross-linking between cell wall polymers in the central cylinder. The results of measurement of the swelling coefficient of standardized cell wall samples in water and in 10 mM KCl at different pH values show that the swelling capacity of root cell walls varies according to the physicochemical properties of synthetic ion exchangers. Cell walls shrink (cell wall volume decreases) as ion concentration in solution increases and pH decreases. This causes an increase in the hydraulic resistance (or a decrease in the hydraulic conductivity) of apoplast. It was concluded that swelling is determined by the physicochemical properties of the cell wall, whereas the change in the swelling capacity induced by variation of external or internal conditions is an element of the mechanism of regulation of volume water flow in roots.     

Mineral composition of Halimione portulacoides (L.) Aellen leaves

Mineral composition ofHalimione portulacoides (L.) Aellen leaves was studied. The leaves accumulate salt, which is partly compensated by succulence. The ratio K/Na is less than 1. No distinct correlation between salt content in the soil and the mineral content at different stations could be found. Communication no. 148 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands.     

Field Assessment of Outcrossing from Transgenic Pea (Pisum Sativum L.) Plants

The frequency of outcrossing from a transgenic line of peas into three cultivars ('Carneval', 'Montana', 'Tipu') was studied in the field in 1997 and 1999. Two dominant traits, normal leaf form and a highly-expressed -glucuronidase (gusA) gene, were used as markers of pollen transfer. Because of heterogeneity in the commercial seed sources, leaf form alone was unreliable for assessing pollen migration into 'trap' plants. Of approximately 9000 offspring tested, only five plants scored positive for the presence of both markers. All five were located in 'trap' plots situated near the transgenic line. This represents a mean outcrossing rate of 0.07%.     

RETRACTED ARTICLE: Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na⁺/H⁺ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na⁺/H⁺ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K⁺, Ca²⁺ and Mg²⁺ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na⁺/H⁺ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work.     

Seasonal fluctuations of the mineral concentration of alder (Alnus glutinosa (L.) Gaertn.) from the field

Summary The seasonal fluctuation of N, P, K, Ca, Mg, Fe, Mn, Mo, and Co, in leaves, roots and nodules of 40–50 year oldAlnus glutinosa trees growing at four different locations along the banks of the Tormes river, in the province of Salamanca, was studied. Also, the evolution of the soil organic matter under the trees sampled was evaluated. The data obtained for the various nutrient elements in the three plant parts are statistically treated at the significance levels of 99–95 per cent, and some remarks as to the nutritional status of the European alder in respect to the nutrients and its contribution to soil nutrient-cycling are provided. A positive correlation was found between N–P, N–K, N–Mg, and N–Mo, in leaves, and between N–P, N–K, N–Fe, N–Mn, and N–Mo in root nodules. In roots only, no significance at any level was obtained between N and any of the elements analyzed.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.     

Active chitinases in the apoplastic fluids of healthy white lupin (Lupinus albus L.) plants

Acidic exocellular class III chitinase (EC 3.2.1.14) was previously identified in healthy white lupin (Lupinus albus L.) plants and suspension-cultured cells by N-terminal microse-quencing. In this study, the detection of chitinase activity with Remazol Brilliant Violet 5R (RBV)-labelled chitin derivatives is described. Chitinase activity was observed in protein fractions of cytoplasmic or exocellular origin from roots, hypocotyls, cotyledons, and leaves of healthy white lupin plants. Using isoelectrofocusing followed by a new overlay technique with carboxymethyl chitin-RBV conjugate-containing gel, up to six different chitinase isoforms were visualised. Their activity was distributed fairly evenly within a plant with acidic isoforms predominating in cell walls and basic (or neutral) ones found intracellularly. Exocellular location of some chitinase isoforms were also confirmed by detection of their activities in intercellular washing fluids from white lupin tissues. Chitinase activity was demonstrated in culture filtrates and cell walls of suspension-cultured white lupin cells.     

Production and Characterization of a Continuous Embryonic Cell Line from Sea Bass (Dicentrarchus labrax L.)

Continuous cell lines represent an important tool both for biological studies and for their applications in marine biotechnology. In this article we describe the production and characterization of a continuous adherent cell line, named DLEC, derived from early embryos of the European sea bass Dicentrarchus labrax L. (Actinopterygii, Moronidae). Cells were obtained by disrupting 2- to 12-hour-old embryos and culturing resulting cells at 18°C in RPMI medium containing 5% fetal calf serum (FCS) and 10% supernatant fraction of the embryo homogenate. After 8 weeks culture medium was replaced with Liebovitz's L15 medium containing 10% FCS and DLEC cells started proliferation. Subsequently, they were continuously cultured until the 50th passage without evident changes in their morphology. DLEC cells show a fibroblast-like shape and a modal chromosome number of 48, as do the wild-type cells; conversely the constant presence of six to nine meta-submetacentric elements in the karyotype (vs. zero to two in the wild-type) indicates the occurrence of chromosomal rearrangements during stabilization. DLEC cells are sensitive to substances known to induce differentiation of mammalian cells such as retinoic acid and phorbol esters. They have been transfected using liposomes with a commercial plasmid vector containing a reporter gene, thus suggesting a possible importance as an alternative expression system of recombinant vertebrate proteins in teleost cells.