A. V. Babakov

Involvement of 14-3-3 proteins in the osmotic regulation of H+- ATPase in plant plasma membranes.

Abstract. Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H+ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes; the 14-3-3 content in the membranes increased 2- to 3-fold, while the H+-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H+-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H+ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins.

Transformation of tobacco and Arabidopsis plants with Stellaria media genes encoding novel hevein-like peptides increases their resistance to fungal pathogens.

Two novel antifungal hevein-like peptides, SmAMP1.1a and SmAMP2.2a, were previously isolated from seeds of Stellaria media. It has been established that these peptides accumulate in this weed as a result of proteolysis of two propeptides, pro-SmAMP1 and pro-SmAMP2. The primary structure of these propeptides is unique; in addition to having a signal peptide and negatively charged C-terminus, each of these structures consists of two hevein-like peptides of different length separated by a space rather than a single peptide. In this work, we demonstrated that the expression of the pro-SmAMP1 and pro-SmAMP2 genes was tissue-specific and increased substantially under exposure to fungal infection. To elucidate whether S. media has any advantages in defending against phytopathogens due to its unusual structure of pro-SmAMP1 and pro-SmAMP2, on the basis of the pro-SmAMP1 gene, we created three genetic constructs. Arabidopsis and tobacco plants were subsequently transformed with these constructs. Transgenic plants bearing the full-length pro-SmAMP1 gene exhibited the best resistance to the phytopathogens Bipolaris sorokiniana and Thielaviopsis basicola. The resistance of S. media plants to phytopathogenic fungi was likely due to the fungal-inducible expression of pro-SmAMP1 and pro-SmAMP2 genes, and due to the specific features of the primary structure of the corresponding propeptides. As a result of the processing of these propeptides, two different antimicrobial peptides were released simultaneously. Based on our results, we conclude that the genes for antimicrobial peptides from S. media may be promising genetic tools for the improvement of plant resistance to fungal diseases.

Purification and subunit composition of a GTP-binding protein from maize root plasma membranes

When frozen plasma membranes isolated from maize seedling roots are thawed, a significant portion of GTP‐binding activity goes into solution. The GTP‐binding protein was purified by ion exchange chromatography on Mono‐Q and gel filtration on Superose 6. Its molecular weight was estimated at 61 kDa by gel filtration. The same molecular weight was obtained upon solubilization of the GTP‐binding protein with cholic acid followed by gel filtration in the presence of this detergent. SDS‐PAGE demonstrated that the isolated GTP‐binding protein consists of two types of subunit of molecular weights 27 kDa and 34 kDa.

Potato plants bearing a vacuolar Na + /H + antiporter HvNHX2 from barley are characterized by improved salt tolerance

Two cultivars of potato (Solanum tuberosum L.) were transformed with a barley antiporter gene HvNHX2 driven by the CaMV 35S promoter. The expressed transgene conferred a higher NaCl tolerance to one of the cultivars. Under salt stress, the more salt-tolerant transgenic plants had longer roots, higher dry weight, and suppressed cell expansion as compared to wild-type plants. The salt tolerance of the plants grown in vitro was not accompanied by elevated total sodium in any plant organs tested. Instead, higher potassium was found in roots of transgenic plants. Possible mechanisms of plant salt tolerance are discussed.

Culture of transformed horseradish roots as a source of fusicoccin-like ligands

Endogenous fusicoccin (FC) or related substances were sought in horseradish (Armoracia rusticana P.) roots. An actively growing root culture was derived from plants transformed with Agrobacterium rhizogenes. The presence of FC-like substances in ethanolic extracts from roots was established in a radioreceptor binding assay with plasmalemmal FC receptors and in radioimmune analysis with an antiserum specific for FC A. FC-like ligands were found in the tissue and medium of aseptically grown culture.

Activity of Ion Transporters and Salt Tolerance in Barley

The authors attempted to relate the cultivar-specific salt tolerance in barley (Hordeum distichum L.) to the efficiency of ion transporters in the plasmalemma and tonoplast. The study involved plasmalemma and tonoplast membrane vesicles isolated from roots and leaves of the 7-day-old barley seedlings exposed to elevated NaCl concentrations. Two barley cultivars were employed: salt-tolerant cv. Elo and salt-susceptible cv. Belogorskii. The vesicles were used to measure the transport activity of plasmalemma and tonoplast proton pumps and the cation/anion exchange. The data obtained in the experiments demonstrated that the changes in the activity of ion transporters under salt stress conditions correlated with the barley cultivar-specific tolerance to elevated NaCl concentrations.

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na+/H+-antiporter and 22Na+ accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study 22Na+ uptake, expression of three isoforms of the Na+/H+ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of 22Na+. However, after 24-h incubation the content of 22Na+ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of 22Na+ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na+ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.

Identification of K+/H+ antiporter homolog in barley: Expression in cultivars with different tolerance to NaCl

A cDNA fragment comprising 80% of complete cDNA nucleotide sequence was identified in barley (Hordeum vulgare L.); it encoded a polypeptide of 401 amino acids and had a high (83%) homology to the tomato K+/H+antiporter, LeNHX2. We supposed that the identified cDNA fragment encodes a new isoform of Na+,K+/H++ antiporter and designated it HvNHX4. The transcripts of the HvNHX4 gene were detected in roots and leaves of barley seedlings; their content did not change under salt stress conditions. Antibodies to C-terminal fragment of HvNHX4 were raised. Western blot analysis of membrane proteins showed that HvNHX4 was located in the vacuolar membranes and probably in the endoplasmic reticulum and Golgi membranes; tracing amounts of this protein were present in plasma membranes. A comparative Western blot analysis was performed for two isoforms (HvNHX4 and HvNHX2) in vacuolar membranes isolated from roots and leaves of four barley cultivars with different resistances to salt stress. The HvNHX4 protein was detected in roots of a salt-susceptible cv. Belogorskii grown under control conditions; its content in roots of a salt-tolerant cv. Moskovskii 121 was found to increase after salt stress. The content of HvNHX2 protein in roots and leaves increased during salt stress in the salt-tolerant cv. Elo but did not change in the susceptible cv. Belogorskii. The results provide evidence for the organ-and cultivar-specific expression of different isoforms of Na+,K+/H+ antiporter in barley and indicate that the expression of these isoforms is regulated at the posttranscriptional level during salt stress.

[Cold shock domain proteins in the extremophyte Thellungiella salsuginea (salt cress): gene structure and differential response to cold].

Four genes encoding cold shock domain (CSD) proteins have been identified in salt cress Thellungiella salsuginea (halophila), an extremophyte currently recognized as a promising model for studying stress tolerance]. The deduced proteins prove highly homologous to those of Arabidopsis thaliana (up to 95% identity) and are accordingly enumerated TsCSDP1-TsCSDP4; after the N-proximal conserved CSD, they have respectively 6, 2, 7, and 2 zinc finger motifs evenly spaced by Gly-rich stretches. Much lower similarity (∼45%) is observed in the regions upstream of TATA-box promoters of TsCSDP1 vs. AtCSP1, with numerous distinctions in the sets of identifiable cis-regulatory elements. Plasmid expression of TsCSDP1 (like AtCSP1/3) rescues a cold-sensitive csp-lacking mutant of Escherichia coli, confirming that the protein is functional. In leaves of salt cress plants under normal conditions, the mRNA levels for the four TsCSDPs relate as 10: 27: 1: 31. Chilling to 4°C markedly alters the gene expression; the 4-day dynamics are different for all four genes and quite dissimilar from those reported for their Arabidopsis homologous under comparable conditions. Thus, the much greater cold hardiness of Thellungiella vs. Arabidopsis cannot be explained by structural distinctions of its CSDPs, but rather may be due to expedient regulation of their expression at low temperature.

Characteristics of Salt-Tolerant and Salt-Susceptible Cultivars of Barley

Twelve cultivars of barley (Hordeum vulgare L.) from the collection of the Vavilov Institute of Plant Industry, Russian Academy of Agricultural Sciences, were screened by assessing the length of 6-day-old seedlings grown in water culture at 70, 120, and 170 mM NaCl. As a result, two salt-susceptible cultivars, Belogorskii and QB 60.1, and three salt-tolerant cultivars, Elo, Odesskii 115, and Local from Ecuador, were selected, and these cultivars were used in the greenhouse soil-culture experiments. The grain yield of salt-tolerant cultivars was affected by NaCl to a lesser degree than that of the salt-susceptible cultivars. In both cases, soil salinization increased the sodium content in the seedlings as compared to the control plants. Characteristically, salt-susceptible cultivars accumulated more Na+ in their shoots than salt-tolerant cultivars; the reciprocal pattern was found in the roots. Soil salinization decreased K+ content in the shoots of the salt-susceptible cv. Belogorskii as compared to the control, whereas in the most tolerant cv. Local from Ecuador, the potassium content increased.