Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures

To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo [35S]methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)- mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity. Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells. Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG). The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly. In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells. These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation. A further group of proteins included those induced after long exposure to both water stress and shock. Western blot analysis revealed that osmotin was one protein belonging to this common group. This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged stress.     

Gene expression associated with water-stress adaptation of rice cells and identification of two genes as hsp 70 and ubiquitin

We have isolated polyethylene glycol (PEG)-adapted rice cells that can proliferate in a medium with 20% PEG as well as in a medium with 1% NaCl. The adapted-cells overproduce a set of proteins whose roles may be associated with water-stress adaptation. To isolate genes encoding these proteins, we differentially screened a cDNA library and obtained 5 cDNA clones which showed preferential hybridization to mRNA of adapted cells. The present paper describes the pattern of expression and the sequence analysis of these 5 genes. Sequence analysis of partial cDNAs indicates that two genes encode the 70 kDa heat shock protein and the ubiquitin but the identities of the other 3 are not known. The expression of all 5 genes fluctuates slightly during the growth cycle of the PEG-adapted cells grown in the control or in the PEG-containing medium. In contrast, gene expression in the parental cells fluctuates to a much greater extent and always begins with an enhanced expression during the first day after subculture. Sub-lethal concentrations of PEG or NaCl have no immediate effect on gene expression in parental cells but NaCl may have a long term effect in enhancing the expression of two genes after 5 days. Abscisic acid (ABA) has no effect on the expression of 4 genes but suppresses the expression of one gene. The roles of these genes in water-stress adaptation of plant cells are discussed.     

Responses to Water Stress in Adapted and Unadapted Carrot Cell Suspension Cultures

The responses of suspension-cultured cells of carrot to polyethyleneglycol (PEG)-induced water stress were studied after transferto culture medium containing PEG at concentrations between 0%and 25%. Growth characteristics, cellular osmotic potentialand organic solute concentration changes were followed in unadaptedcells and in cell lines adapted to growth in various PEG concentrations.A decline in fresh and dry weight increase occurred in unadaptedcells with decreasing water potential, while dry weight gainwas unaffected in adapted lines. Substantial osmotic adjustmentwas observed in adapted lines, due mainly to increased glucose,fructose and sucrose. Proline concentration increased up to40-fold in adapted and 12-fold in unadapted cells and otheramino acids including alanine, histidine and arginine showedsimilar, though smaller, responses. Polyamines and glycinebetainedid not increase significantly in either adapted or unadaptedcells. Changes leading to long-term adaptation to water stressare discussed in relation to short-term stress—shock responses. Key words: Water stress, cell culture, Daucus carota, osmotic adaptation, solute accumulation     

Initiation and regulation of water deficit-induced abscisic acid accumulation in maize leaves and roots: cellular volume and water relations

Water deficit-induced ABA accumulation in relation to cellular water relations was investigated in maize root and leaf tissues. While polyethylene glycol (PEG) treatment led to a significant increase of ABA content in both root and leaf tissues, ethylene glycol (EG), a permeable monomer of PEG, had no effect on ABA accumulation at similar or much lower osmotic potentials. A rapid and massive accumulation of ABA in leaf tissues occurred at a specific threshold of PEG 6000 concentration, about 20% (w/v), and closely coincided with the start of the tissue weight loss and the obvious decrease of cellular osmotic potential. Pretreatment with EG lowered the cell sap osmotic potential and also lowered the capability of both root and leaf tissues to accumulate ABA in response to further air-drying or PEG treatment. When samples were dehydrated and incubated under pressure, a method to maintain high water potential and pressure potential during dehydration, ABA accumulation was similar to those dehydrated and incubated under atmospheric pressure. Such results suggest that both the absolute water potential and pressure potential per se had no direct effects on the dehydration-induced ABA accumulation. The results have provided evidence that the initiation of ABA accumulation is related to the weight loss of tissues or changes in cellular volume rather than the cell water relation parameters, and the capability of ABA accumulation can be regulated by cellular osmotic potential.     

Study on the Heat Shock Protein-Hpc60 from Pea Leaf Cytosol

The effects of NaC1, PEG and exogenous ABA on the expression of the heat shock protein-Hpc60 from pea ( Pisum sativum L. ) were investigated. The results showed that PEG elevated the quantity of this heat-shock protein, much similar to heat-shock stress. Although NaC1 was able to reduce water potential at the same extent with or even higher than PEG, but it did not increase the level of this protein. This phenomenon was closely correlated with the variations of cytoplasmic ion levels. The authors postulated that NaC1 treatment caused the influx of Na + , thus disrupted the ion homeostasis, whereas PEG treatment did not. So a rather stable Na+/K+ ratio was sustained in the plant cell treated with PEG, which might be prerequisite for the induction of Hpc60. In addition, exogenous ABA was employed in the authors' experiment which showed no influence on the synthesis of this heat-shock protein.     

Acclimation of potato plants to polyethylene glycol-induced water deficit I. Photosynthesis and metabolism

The acclimation of photosynthesis and metabolism in response to water deficit is characterized using hydroponically grown potato plants (Solanum tuberosum cv. Désirée). Plants were subjected to a reduced water potential of the nutrient solution by adding 10% (w/v) PEG 6000. PEG-treated plants were retarded in growth. Leaves which had been fully developed before the PEG treatment and leaves grown during the PEG treatment showed different phenotypes and biochemical and physiological properties. Photosynthesis of all leaves decreased during the whole treatment. However, the decrease of photosynthesis in the two types of leaves had different causes indicated by differences in their metabolism. Leaves which were fully developed at the beginning of the PEG treatment began to wilt starting from the leaf rim. The apoplastic ABA content increased, coinciding with a decreased stomatal conductance. Increased energy charge of the cells indicated impaired chloroplastic metabolism, accompanied by a decrease of amounts of chloroplastic enzymes. The apoplastic and the symplastic ABA content were increased during water deficit and because ABA was concentrated in the cytosolic compartment it is suggested that ABA is involved in decreasing photosynthetic enzyme contents in old leaves. Young leaves, grown after the imposition of water deficit, were smaller than control leaves and had a curly surface. In young leaves apoplastic and cytosolic ABA contents were identical with control values. Carboxylation efficiency of photosynthesis was decreased, but the water use efficiency remained unchanged. Metabolic data of the photosynthetic pathways indicate a down-regulation of chloroplastic metabolism. It is concluded that in young leaves photosynthesis was non-stomatally limited. This limitation was not caused by ABA.     

A hypocotyl-derived somatic embryogenic system in Brassica juncea Czern & Coss and its manipulation for enhanced storage lipid accumulation

A simple and reproducible protocol for induction, growth and development of somatic embryos from hypocotyl explants of Indian mustard (Brassica juncea L. Czern & Coss) var. RLM 198 is reported. The HDSE (Hypocotyl-derived somatic embryos) were fleshy globular to torpedo structures that were maintained by regular subculturing every three weeks. These embryos developed non-synchronously into the heart shaped-stage while some were matured into a green cotyledon-stage bearing embryos in the same medium. The HDSE accumulated as much as 50.2% lipid content on a dry weight basis at 14 DAC (days after culture) using a culture medium supplemented with 10% PEG (Polyethylene glycol 6000) in comparison to less than 15% lipid content in 2% sucrose (control) or 20 μM ABA (abscisic acid). An increase in total soluble sugar content was observed with 2.5% PEG and increasing PEG concentration caused a decrease in their contents in HDSE. The activities of invertase, acetyl CoA carboxylase and 1-¹⁴C-acetate incorporation into lipids in HDSE were enhanced significantly in the culture medium containing 10% PEG. The content of triacylglycerols in HDSE was maximum with 10% PEG supplemented culture medium. The wax content in HDSE increased progressively with an increase of PEG concentration in the culture medium. The ABA and PEG supplementation increased the content of membrane lipids when the data was expressed on a 100 g dry weight basis. The proportion of palmitate and erucate decreased and that of oleate, linoleate and linolenate increased at 14 DAC in HDSE in 10% PEG supplemented culture medium. Thus, the manipulation of culture conditions significantly altered total lipid content, membrane lipid composition and the quality of storage lipids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Hydraulic and Chemical Responses of Citrus Seedlings to Drought and Osmotic Stress

In this work we investigated the function of abscisic acid (ABA) as a long-distance chemical signal communicating water shortage from the root to the shoot in citrus plants. Experiments indicated that stomatal conductance, transpiration rates, and leaf water potential decline progressively with drought. ABA content in roots, leaves, and xylem sap was also increased by the drought stress treatment three- to sevenfold. The addition of norflurazon, an inhibitor of ABA biosynthesis, significantly decreased the intensity of the responses and reduced ABA content in roots and xylem fluid, but not in leaves. Polyethylene glycol (PEG)-induced osmotic stress caused similar effects and, in general, was counteracted only by norflurazon at the lowest concentration (10%). Partial defoliation was able to diminish only leaf ABA content (22.5%) at the highest PEG concentration (30%), probably through a reduction of the active sites of biosynthesis. At least under moderate drought (3–6 days without irrigation), mechanisms other than leaf ABA concentration were required to explain stomatal closure in response to limited soil water supply. Measurements of xylem sap pH revealed a progressive alkalinization through the drought condition (6.4 vs. 7.1), that was not counteracted with the addition of norflurazon. Moreover, in vitro treatment of detached leaves with buffers iso-osmotically adjusted at pH 7.1 significantly decreased stomatal conductance (more than 30%) as much as 70% when supplemented with ABA. Taken together, our results suggest that increased pH generated in drought-stressed roots is transmitted by the xylem sap to the leaves, triggering reductions in shoot water loss. The parallel rise in ABA concentration may act synergistically with pH alkalinization in xylem sap, with an initial response generated from the roots and further promotion by the stressed leaves.     

Influence of Osmotic Stress on Nitrate Reductase Activity in Wheat (Triticum aestivum L.) and the Role of Abscisic Acid

Wheat seedlings (Triticum aestivum L. cv. Timmo) were treatedwith up to 20% (w/v) polyethylene glycol (PEG, mol. wt. 3350)in the nutrient medium for 6 d. Shoot growth and nitrate transportand metabolism were substantially affected by PEG treatment.At 20% PEG (corresponding to a water potential of approximately–1.6 MPa), which caused plants to wilt within 1–2h, activity of nitrate reductase (NR) declined with a half-lifeof approximately 5 h in both roots and shoots. The decline wasconsiderably slower at lower PEG concentrations. Significantincreases in levels of abscisic acid (ABA) only occurred inshoots. Application of ABA to intact plants or excised shootsdid not induce or accelerate decline in shoot NR activity. Therapid decline in NR activity during wilting appears unrelatedto both nitrate flux and ABA. At lower PEG concentrations andin the long-term, however, NR activity corroborates rates ofboth transport and growth-related utilization of nitrate. Therole of ABA in this context appears to be indirect through itsaction on stomatal function which reduces water flux and gasexchange. Key words: Stress, nitrate reductase, abscisic acid (ABA)     

Somatic Embryogenesis in Abies Alba × Abies Alba and Abies Alba × Abies Nordmanniana Hybrids

Maturation and germination of somatic embryos of hybrids A. alba × A. alba and A. alba × A. nordmanniana were followed by protein analysis of single embryogenic -suspensor masses (ESM) and analysis of storage protein accumulation during somatic embryo development. Very important step was one week pre-cultivation of ESM on medium with polyethylene glycol (PEG) and abscisic acid (ABA). Low osmotic potential of maturation medium and addition of ABA supported development of somatic embryo. Also partial drying of somatic embryo during following three weeks was needed for its normal development. In spite of morphologically fully developed, the somatic embryos were not physiologically ready for germination at least in terms of storage protein accumulation.     

Abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato

Summary Somatic embryos of sweet potato have potential as synthetic seeds. The effects of abscisic acid (ABA) (0,0,0.1, 1.0, 10.0 and 50.0 μM) were examined to improve synchrony and proliferation of somatic embryos. Transferring embryos compared to those cultures transferred at day 0. The development of embryos in suspension culture supplemented with ABA was poor. However, when calli proliferation cultures were in gelled medium and pulsed with 0.1 μM ABA for 14 d, the number of somatic embryos increased. Proembryonic masses cultured in mannitol-containing medium (Y=−1.5 MPa) increased embryo development and synchrony of embryo development. Thus, in this work ABA and mannitol have been shown to improve both the total number and the synchrony of sweet potato somatic embryos.     

Long-term somatic embryogenesis and maturation of somatic embryos in Hevea brasiliensis

A high frequency of secondary embryogenesis was induced from isolated early cotyledonary-stage somatic embryos of Hevea brasiliensis. A long-term embryogenic line was established by the use of recurrent embryogenesis and maintained for 3 years on hormone-free medium by the transfer of selected proembryogenic masses every 10 days.

The addition of 234 mM sucrose as stress with sucrose and 10⁻⁵ M abscisic acid (ABA) to the culture medium enhanced the maturation of somatic embryos. Under these culture conditions, the embryo population was composed of 45% globular, 18% oblong and 37% torpedo-stage embryos. These somatic embryos had well-formed tissue structure, a well-defined epidermis, protein storage bodies, and a high accumulation of starch. The triglyceride content was five times as high in the torpedo-stage embryos that developed on medium supplemented with 234 mM sucrose and 10⁻⁵ M ABA as in embryos obtained on basal medium with 58 mM sucrose.     

Relations between MAPK phosphorylation and ABA level in <Emphasis Type="Italic">Malus sieversii</Emphasis> under water stress

A correlation between protein kinase phosphorylation and ABA level was studied in Malus sieversii (Ledeb.) Roem. seedlings under water stress. The seedlings were treated with PEG 6000 for imitation of water stress, and the MAPK activity and ABA content in each treatment were then determined. We demonstrated that the increase in the activities of the total protein kinase (TPK) and mitogen-activated protein kinase (MAPK) after treatment with 20% PEG 6000 appeared to result in a high level of ABA. MAPK activity accounted for 76.8% of TPK activity. The activity peaks of TPK and MAPK preceded the highest level of ABA accumulation. It is interesting that the ABA level in roots and leaves of seedlings pretreated with 2 × 10⁻² mM exogenous ABA for 20 min following treatment by 20% PEG 6000 was much higher than that of seedlings treated with exogenous ABA only. We analyzed the influence of MAPK inhibitor ITU (5-iodotubercidin) on ABA accumulation in the seedlings of M. sieversii under water stress and showed that 1 μM ITU significantly decreased the ABA level induced by a water loss. However, the phosphoesterase inhibitor PAO (phenylarisine oxide) enhanced ABA accumulation, indicating that the phosphorylated MAPK was correlated to ABA synthesis. Together, these results suggest that MAPK phosphorylation played an important role in ABA accumulation under water stress, and MAPK might mediate the signal transduction of ABA synthesis.     

Differential effects of permeating and nonpermeating solutes on the fatty acid composition of Pseudomonas putida

We examined the effect of reduced water availability on the fatty acid composition of Pseudomonas putida strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes NaCl or polyethylene glycol (PEG) with a molecular weight of 200 (PEG 200) or the nonpermeating solute PEG 8000. Transmission electron microscopy showed that -1.0-MPa PEG 8000-treated cells had convoluted outer membranes, whereas -1.0-MPa NaCl-treated or control cells did not. At the range of water potential (-0.25 to -1.5 MPa) that we examined, reduced water availability imposed by PEG 8000, but not by NaCl or PEG 200, significantly altered the amounts of trans and cis isomers of monounsaturated fatty acids that were present in whole-cell fatty acid extracts. Cells grown in basal medium or under the -0.25-MPa water potential imposed by NaCl or PEG 200 had a higher trans:cis ratio than -0.25-MPa PEG 8000-treated cells. As the water potential was lowered further with PEG 8000 amendments, there was an increase in the amount of trans isomers, resulting in a higher trans:cis ratio. Similar results were observed in cells grown physically separated from PEG 8000, indicating that these changes were not due to PEG toxicity. When cells grown in -1.5-MPa PEG 8000 amendments were exposed to a rapid water potential increase of 1.5 MPa or to a thermodynamically equivalent concentration of the permeating solute, NaCl, there was a decrease in the amount of trans fatty acids with a corresponding increase in the cis isomer. The decrease in the trans/cis ratio following hypoosomotic shock did not occur in the presence of the lipid synthesis inhibitor cerulenin or the growth inhibitors chloramphenicol and rifampicin, which indicates a constitutively operating enzyme system. These results indicate that thermodynamically equivalent concentrations of permeating and nonpermeating solutes have unique effects on membrane fatty acid composition.     

Heat shock protein of the Hsp70 family in the euryhaline cilate Paramecium nephridiatum and its role in adaptation to salinity changes

The level of the Hsp70 heat shock protein was studied in the cells of euryhaline ciliates Paramecium nephridiatum after environmental salinity changes. Two types of treatment were used: “shock” and “adaptation.” In the former case the ciliates were placed for 1 h into medium with stress salinity, and subsequently returned for 2 h to the medium they were acclimated to. In the latter case the ciliates were placed for 3 h into the medium with the stress level of salinity. The ciliates acclimated to fresh water (0‰ salinity) were shown to have a higher constitutive level of Hsp70 than those acclimated to 10‰. Transfer of the protists from fresh water to medium with 10‰ salinity (the shock medium) did not increase Hsp70 synthesis, whereas the return transfer resulted in induction of Hsp70 in the cells. In both directions of salinity change, “adaptation” led to induction of Hsp70. The obtained results support the hypothesis that salinity of 10‰ is less harmful for the eurihaline ciliate P. nephridiatum, than fresh water is. We also presume that the ability of euryhaline ciliates to survive in a wide salinity spectrum might be determined by the higher constitutive level of their Hsp70 in comparison with that of stenohaline representatives of the same genus.     

The relationship between stomatal resistance and abscisic-acid levels in leaves of water-stressed bean plants

Leaf water potentials of Phaseolus vulgaris L. plants exposed to a -3.0 bar root medium were reduced to between -7 and -9 bars within 25 min and remained constant for the next several hours. This treatment led to considerable variation between leaves in both abscisic-acid (ABA) content and R_s, although the two were well correlated after a 5-h treatment. There was an apparent 7-fold increase in leaf ABA levels necessary to initiate stomatal closure when plants were exposed to a -3.0 bar treatment, but when plants were exposed to a -5.0 bar stress R_s values increased prior to any detectable rise in ABA levels. To explain these seemingly contradictory results, we suggest that the rate of ABA synthesis in the leaf, rather than the total ABA content, determines the status of the stomatal aperture.Abbreviations ABA abscisic acid - PEG polyethylene glycol - R_s stomatal diffusion resistance of lower leaf surface - leaf water potential     

Does abscisic acid influence proline accumulation in stressed leaves?

Root treatments of barley (Hordeum distichum L.) plants with 10^-7 to 10^-4 M abscisic acid (ABA) caused an increase in proline content, especially at higher concentrations, within 2–3 h. Even 3 h after the removal of ABA from the medium the plants continued to accumulate proline. The higher the concentration of the ABA, the higher was the proline level at 6 h. When the highest ABA concentration, 10^-4 M, was tested with polyethylene glycol (PEG) (-5.0 bars) in the medium, the ABA treatment resulted in a higher proline content than in control plants. The treatments PEG alone and PEG + ABA resulted in heavy accumulation of proline, especially, 3 h after releasing the plants from the stress. The proline content in PEG+ABA-treated plants was always higher than plants treated with PGE or ABA alone. In peas (Pisum sativum L. cv Alaska) the same trend occurred although to a lesser degree. These findings indicate an influence of ABA on proline accumulation in water-stressed plants.Abbreviations ABA abscisic acid - PEG polyethylene glycol - RWC relative water content     

Acclimation to low water potential in potato cell suspension cultures leads to changes in putrescine metabolism

Changes in levels and biosynthesis of di- and polyamines are associated with stress responses in plant cells. The involvement of these molecules was investigated here in cultured potato (Solanum tuberosum L.) cells grown in medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin, and acclimated or not to low water potential. The diamine (putrescine) and polyamine (spermidine and spermine) status in cells gradually acclimated to increasing concentrations (up to 20 %, w/v) of polyethylene glycol (PEG) Mr 8000, was compared with that of unacclimated cells abruptly exposed (shocked) or not (controls) to 20 % (w/v) PEG. After a 72-h subculture, the free and perchloric acid (PCA)-soluble conjugated di- and polyamine pattern in acclimated cells was not dramatically different from that of controls, but PCA-insoluble conjugated putrescine was 14-fold higher than in controls. In shocked cells, a strong reduction in free putrescine and spermidine/spermine titres occurred. Arginine (ADC, EC 4.1.1.19) and ornithine (ODC, EC 4.1.1.17) decarboxylase activities were not substantially altered in shocked cells compared with controls, while in PEG-acclimated cell populations they increased about 3-fold, both in the soluble and particulate fractions. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) and diamine oxidase (DAO, EC 1.4.3.6) activities followed a similar pattern to each other in that their activities were enhanced 2- and 3-fold, respectively, in acclimated cells over unacclimated controls. Ethylene production was also enhanced in acclimated cells. These results indicate that, with respect to di- and polyamines, acquired tolerance to low water potential in potato cells leads principally to changes in putrescine biosynthesis and conjugation which may be involved in ensuring cell survival.