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The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germination, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant-specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co-immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.

Summary Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxial–abaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5′-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxial–abaxial polarity.

Sumoylation is a post-translational regulatory process in diverse cellular processes in eukaryotes, involving conjugation/deconjugation of small ubiquitin-like modifier (SUMO) proteins to other proteins thus modifying their function. The PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. Here, we report the isolation and molecular characterization of Oryza sativa SIZ1 (OsSIZ1) and SIZ2 (OsSIZ2), rice homologs of Arabidopsis SIZ1. The rice SIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Our analysis showed increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABA-hypersensitivity of siz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation.

*† These authors contributed equally to this work. SUMMARY In this study, the meiotic role of MEIOTIC CONTROL OF CROSSOVERS1 (MCC1), a GCN5-related histone N-acetyltransferase, is described in Arabidopsis. Analysis of the over-expression mutant obtained by enhancer activation tagging revealed that acetylation of histone H3 increased in male prophase I. MCC1 appeared to be required in meiosis for normal chiasma number and distribution and for chromosome segregation. Overall, elevated MCC1 did not affect crossover number per cell, but has a differential effect on individual chromosomes elevating COs for chromosome 4, in which there is also a shift in chiasma distribution, and reducing COs for chromosome 1 and 2. For the latter there is a loss of the obligate CO/chiasma in 8% of the male meiocytes. The meiotic defects led to abortion in about half of the male and female gametes in the mutant. In wild type, the treatment with trichostatin A, an inhibitor of histone deacetylases, phenocopies MCC1 over-expression in meiosis. Our results provide evidence that histone hyperacetylation has a significant impact on the plant meiosis.

Ledidi, Eduardo O. et al.-- 12 páginas, 6 figuras, 3 tablas., NHX-type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na+ in vacuoles. However, all isoforms characterized so far catalyze both Na+/H+ and K+/H+ exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K+, which in turn affects plant K+ nutrition and Na+ tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K+ vacuolar pools in all growth conditions tested, but no consistent enhancement of Na+ accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K+ and to withstand salt-shock. Under K+-limiting conditions, greater K+ compartmentation in the vacuole occurred at the expense of the cytosolic K+ pool, which was lower in transgenic plants. This caused the early activation of the high-affinity K+ uptake system, enhanced K+ uptake by roots, and increased the K+ content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H+-linked K+ transport that is thought to facilitate active K+ uptake at the tonoplast, and the partitioning of K+ between vacuole and cytosol., This work was supported by grants BFU2006-06968 from Ministerio de Educación y Ciencia and AGR-1482 from Junta de Andalucíia to JMP. Additional support was obtained from CSIC-CONACYT Bilateral Cooperation Grant 2004MX0021.

*Summary Salt Overly Sensitive 1 (SOS1), a plasma membrane Na+ /H + antiporter in Arabidopsis, is a salt tolerance determinant crucial for the maintenance of ion homeostasis in saline stress conditions. SOS1 mRNA is unstable at normal growth conditions, but its stability is substantially increased under salt stress and other ionic and dehydration stresses. In addition, H 2O2 treatment increases the stability of SOS1 mRNA. SOS1 mRNA is inherently unstable and rapidly degraded with a half-life of approximately 10 min. Rapid decay of SOS1 mRNA requires new protein synthesis. Stress-induced SOS1 mRNA stability is mediated by reactive oxygen species (ROS). NADPH oxidase is also involved in the upregulation of SOS1 mRNA stability, presumably through the control of extracellular ROS production. The cis-element required for SOS1 mRNA instability resides in the 500-bp region within the 2.2 kb at the 3¢ end of the SOS1 mRNA. Furthermore, mutations in the SOS1 gene render sos1 mutants more tolerant to paraquat, a non-selective herbicide causing oxidative stress, indicating that SOS1 plays negative roles in tolerance of oxidative stress. A hypothetical model for the signaling pathway involving SOS1-mediated pH changes, NADPH oxidase activation, apoplastic ROS production and downstream signaling transduction is proposed, and the biological significance of ROS-mediated induction of SOS1 mRNA stability is discussed.

The plasma membrane Na + /H + -antiporter salt overly sensitive1 (SOS1) from the halophytic Arabidopsis-relative Thel- lungiella halophila (ThSOS1) shows conserved sequence and domain structure with the orthologous genes from Arabidopsis thaliana and other plants. When expression of ThSOS1 was reduced by RNA interference (RNAi), pronounced characteristics of salt-sensitivity were observed. We were interested in monitoring altered transcriptional responses between Thellungiella wild type and thsos1-4, a representative RNAi line with particular emphasis on root responses to salt stress at 350 mmol/L NaCl, a concentration that is only moderately stressful for mature wild type plants. Transcript profiling revealed several functional categories of genes that were differently affected in wild-type and RNAi plants. Down-regulation of SOS1 resulted in different gene expression even in the absence of stress. The pattern of gene induction in the RNAi plant under salt stress was similar to that of glycophytic Arabidopsis rather than that of wild type Thellungiella. The RNAi plants failed to down-regulate functions that are normally reduced in wild type Thellungiella upon stress and did not up-regulate functions that characterize the Thellungiella salt stress response. Metabolite changes observed in wild type Thellungiella after salt stress were less pronounced or absent in RNAi plants. Transcript and metabolite behavior suggested SOS1 functions including but also extending its established function as a sodium transporter. The down-regulation of ThSOS1 converted the halophyte Thellungiella into a salt-sensitive plant.

Summary Reversible modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins are involved in many cellular processes in yeast and animals. Yet little is known about the function of sumoylation in plants. Here, we show that the SIZ1 gene, which encodes an Arabidopsis SUMO E3 ligase, regulates innate immunity. Mutant siz1 plants exhibit constitutive systemic-acquired resistance (SAR) characterized by elevated accumulation of salicylic acid (SA), increased expression of pathogenesis-related (PR) genes, and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Transfer of the NahG gene to siz1 plants results in reversal of these phenotypes back to wild-type. Analyses of the double mutants, npr1 siz1, pad4 siz1 and ndr1 siz1 revealed that SIZ1 controls SA signalling. SIZ1 interacts epistatically with PAD4 to regulate PR expression and disease resistance. Consistent with these observations, siz1 plants exhibited enhanced resistance to Pst DC3000 expressing avrRps4, a bacterial avirulence determinant that responds to the EDS1/PAD4-dependent TIR-NBS-type R gene. In contrast, siz1 plants were not resistant to Pst DC3000 expressing avrRpm1, a bacterial avirulence determinant that responds to the NDR1-dependent CCNBS-type R gene. Jasmonic acid (JA)-induced PDF1.2 expression and susceptibility to Botrytis cinerea were unaltered in siz1 plants. Taken together, these results demonstrate that SIZ1 is required for SA and PAD4mediated R gene signalling, which in turn confers innate immunity in Arabidopsis.

However,the precise mechanism by which osmotin interacts withspecific fungal pathogens and mediates plasma membranepermeability has not been clearly elucidated. Recent ge-netic and biochemical data indicate that osmotin utilizes asignal transduction pathway in yeast to increase thesusceptibility of a target fungus to its cytotoxic effects.

Verticillium dahliae Klebahn is a soil-borne fungal pathogen causing vascular diseases. The pathogen penetrates the host through the roots, spreads through the xylem, and systemically colonizes both resistant and susceptible genotypes. To elucidate the genetic and molecular bases of plant-Verticillium interactions, we have developed a pathosystem utilizing Arabidopsis thaliana and an isolate of V. dahliae pathogenic to both cruciferous and non-cruciferous crops. Relative tolerance (based on symptom severity) but no immunity was found in a survey of Arabidopsis ecotypes. Anthocyanin accumulation, stunting, and chlorosis were common symptoms. Specific responses of the more susceptible ecotype Columbia were induction of early flowering and dying. The more tolerant ecotype C-24 was characterized by pathogen-induced delay of transition to flowering and mild chlorosis symptoms. Genetic analysis indicated that a single dominant locus, Verticillium dahliae-tolerance (VET1), likely functioning also as a negative regulator of the transition to flowering, was able to convey increased tolerance. VET1 was mapped on chromosome IV. The differential symptom responses observed between ecotypes were not correlated with different rates of fungal tissue colonization or with differential transcript accumulation of PR-1 and PDF1.2 defense genes whose activation was not detected during the Arabidopsis-V. dahliae interaction. Impairment in salicylic acid (SA)- or jasmonic acid (JA)-dependent signaling did not cause hypersensitivity to the fungal infection, whereas ethylene insensitivity led to reduced chlorosis and ABA deficiency to reduced anthocyanin accumulation. The results of this study clearly indicated that the ability of V. dahliae to induce disease symptoms is also connected to the genetic control of development and life span in Arabidopsis.

*Summary Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 10 10 ‐10 11 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower K i (2.1 pM) than parental scN, due to its increased association rate constant (2.6 6 0.09 3 10 7 M ‐1 sec ‐1 ). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

Summary The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR-5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1-v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1-d allele or a null ssd1Δ mutation displayed high sensitivity to osmotin. The SSD1-v protein mediates osmotin resistance in a cell wall-dependent manner. Deletion of SSD1-v or SSD1-d impeded sorting of the PIR proteins (osmotin-resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post-transcriptional regulation of gene expression. The sensitivity of ssd1Δ cells to osmotin was only partially suppressed by over-accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall-mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell-wall morphology and lower levels of alkali-insoluble cell-wall glucans. Therefore SSD1 is an important regulator of fungal cell-wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR-5 proteins.

9 pages, 6 figures, 1 table, 35 references., Osmotin is a plant PR-5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR-5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an α-1,2-mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6-mannose backbone of the outer chain of cell wall N-linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an α-1,3-mannosyltransferase that adds the terminal mannose to the outer chain branches of N-linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR-5 proteins., This work was supported by a Spanish Government Fellowship (J.I.I.), NSF Award No. 9808551-MCB and USDA Cooperative award No. 58-6435-8-094.

Griffonia simplicifolia leaf lectin II (GS-II) is a N-acetylglucosamine (GlcNAc) binding protein, consisting of large (rGS-II) and small (sGS-II) subunits, that is presumed to function in plant defense because both the holoprotein and the rGS-II inhibited insect growth and development in feeding bioassays. A cDNA encoding sGS-II has been isolated and the deduced peptide has sequence similarity to class III chitinases. However, neither the GS-II holoprotein nor bacterially expressed recombinant sGS-II proteins had detectable chitinase or lysozyme activities. Both rGS-II and sGS-II mRNAs accumulated in response to methyl jasmonate (Me-JA) treatment but not after wounding in local leaves unless injury was followed by treatment with the ethylene action inhibitor norbornadiene (NBD). Salicylic acid (SA) suppressed wounding/NBD induction of GS-II transcript accumulation and ethylene inhibited Me-JA-induced expression. Apparently, defensive gene expression induced by signal transduction through the octadecanoid pathway in local leaves is suppressed by stress induced ethylene that is produced as a consequence of wounding. However, in leaves systemic to the wound site, rGS-II mRNA levels increased substantially indicating that physiological levels of ethylene are insufficient to down regulate defensive gene expression away from the site of injury. It seems that G. simplicifolia has evolved a rather specialized response to herbivore attack whereby local activation of defensive gene expression is attenuated in order to mount a more substantial defense distal to the site of invasion.

Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.

Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings (Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica. The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.

Cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) adapted to NaCI exhibited significant morphological and physiological changes. Adapted cells remained smaller and more isodiametric at maturity than unadapted cells. The vacuole increased in size relative to the cytoplasm and an extensive network of transvacuolar membrane strands developed. These changes altered the surface contact area between the cytoplasm and the vacuole substantially. In addition, the network of Hechtian strands that anchor the cortical structure to the cell wall became more extensively branched possibly facilitating surface contact of the cytoplasm to the extracellular matrix. Many changes in membrane proteins could also be identified after NaCl adaptation. In particular, a 50-kDa protein that is associated with the plasma membrane and tonoplast was induced during adaptation. Immunocytochemical localization indicated that this 50-kDa protein is associated with Golgi vesicles. By immunoscreening using anti-50-kDa antibody, a 1.71-kb cDNA clone (p50C) was isolated from a λ-ZAP cDNA expression library. The sequence of p50C did not show any significant identity with other genes. Because of the very low abundance of the p50C message, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze p50C gene expression. Immunoblot and quantitative RT-PCR analyses indicated that the expression of this gene is regulated developmentally since the mRNA and protein increased with age in salt-adapted cells but decreased with age in unadapted cells. Also in tobacco plants, p50C mRNA was more abundant in younger leaves than in older leaves. The gene was responsive to NaCI in tobacco cells and to ABA in tobacco seedlings.

H6 is an Atriplex nummularia gene having high sequence homology with the algal caltractin (a basal-body-associated calcium-binding protein) gene. Recombinant H6 was expressed at high levels in Escherichia coli. The recombinant protein exhibited an ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid-induced mobility shift during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was able to bind 45 Ca 2+ . An H6 cDNA probe detected three RNA transcripts of 1.3, 2.2 and 2.4 kb, respectively. The levels of these transcripts were regulated by different environmental cues and during developmental stages. The steady state levels of the 1.3-kb mRNA decreased after touch and heat treatments. Expression of the 2.2-kb message correlated with cell proliferation activity. In cultured cells, the highest level of the 2.2-kb message preceeded the peak of mitotic cell division activity. In plants, the 2.2-kb message was detected only in shoot tips that contained meristematic tissues. The 2.4-kb message was detected exclusively in heat-shocked cells. The relationship among the three transcripts is discussed in the context of the possible role of H6 in mediating developmental and environmental signals.

Molecular investigation of the process of cold acclimation in woody plants has been limited by the superimposition of dormancy-related events on the process of cold tolerance development. To address this limitation, we have used the grape Vitis labruscana L. cv. Concord to develop a system in which the developmental programme of dormancy can be induced seperately from cold acclimation. Using this system we have characterized differential accumulation of several proteins in grape buds during the normally superimposed endodormancy and cold acclimation programmes, and in buds which have entered only the endodormancy programme. A set of 47 kD proteins accumulated during endodormancy without cold acclimation to a level similar to that found in endodormant and cold-acclimated buds, but without any associated increase in bud cold-acclimation level. However, a 27 kD LEA-like protein accumulated only in cold acclimated buds. We conclude that expression of the 47 kD glycoprotein is endodormancy-related, but is not strictly related to the development of cold acclimation, while the 27 kD protein appears to be more specific to cold acclimation. In addition to strengthening the association of LEA-like proteins with cold acclimation, this system allows more specific assessment of cold acclimation-associated phenomena in overwintering buds.

Osmotin gene expression and protein synthesis were up-regulated in young tomato (Lycopersicon esculentum cv. Rheinlands Ruhm) plants after a short exposure (24 h) to 150 mM NaCl or 100 μM abscisic acid (ABA) or after dehydration to 80% of original plant fresh weight. Osmotically induced accumulation of osmotin mRNA was accompanied by a large increase in endogenous ABA levels. Increasing accumulation of osmotin protein was also observed during a longer exposure (7 days) to salt. Upon treatment with NaCl. osmotin mRNA levels increased in both root and leaf tissues. with an additional longer transcript induced in roots. No induction of osmotin mRNA was observed upon salt or water stress of the tomato ABA-deficient mutant sitiens. Treatment of sitiens with exogenous ABA induced osmotin mRNA accumulation to the level normally found in salt-treated wild-type plants. However, salt stress alone enhanced accumulation of osmotin mRNA in plants of another tomato mutant (flacca) which is also impaired in ABA synthesis. In tobacco plants carrying an osmotin promoter/β-glucuronidase (GUS) fusion gene, NaCl induction of GUS could be only partially blocked by the ABA inhibitor fluoridone. In flacca plants simultaneous treatment with NaCl and ABA resulted in higher levels of osmotin transcript compared to those following treatment with NaCl alone. No accumulation of osmotin protein was observed after short- or long-term osmotic treatments of the mutants. Our results support previous evidence that osmotin gene expression may be triggered in part through ABA and in part through a separate pathway of gene activation.

A cDNA clone encoding an isoform of the plasma membrane H+-ATPase was isolated from Nicotiana tabacum. The steady-state plasma membrane H+-ATPase message levels were the same in unadapted tobacco cells and tobacco cells adapted to 428 mol m−3 NaCl. When cells adapted to 428 mol m−3 NaCl maintained in the absence of NaCl (deadapted) for an excess of 100 passages were exposed to 400 mol m−3 NaCl for 24 h, there was an increased accumulation of plasma membrane H+-ATPase message. The NaCl responsiveness of the deadapted cells was dependent upon the growth cycle stage. Alterations in the levels of plasma membrane FT-ATPase message during the growth cycle support a role for the H+-ATPase in cell growth. These results document the induction by NaCl of plasma membrane FT-ATPase message accumulation in tobacco cells, and suggest that enhanced expression of the plasma membrane FT-ATPase has a role in the short term response of cells of NaCl, but is not necessarily involved in long-term adaptation.

Cells of tobacco adapted to grow in high concentrations of NaCl develop tight zones of adhesion between the plasma membrane and cell wall, revealed by concave plasmolysis in osmotic solutions. Unadapted cells exhibit mostly convex plasmolysis and exhibit little or no adhesive character. Wall-less protoplasts isolated from the adapted cells retain the complementary adhesive character and adhere tightly to each other, whereas protoplasts from unadapted cells do not. The hexapeptide gly-arg-gly-asp-ser-pro, in which the arg-gly-asp represents the integrin-binding domain of several animal extracellular matrix proteins,specifically blocks adhesion of the protoplasts. A control hexapeptide, gly-arg-gly-glu-ser-pro, is ineffective in blocking adhesion. Tobacco proteins immunologically related to human vitronectin were found in cell walls and membranes of unadapted and NaCI adapted cells, but the total extractable vitronectin-like protein was enriched in the adapted cells. Tobacco proteins immunologically related to human fibronectin were found in membranes and cell walls of NaCI adapted cells but not in those from unadapted cells.Our observations indicate that plant cells possess cell-matrix adhesion complexes similar to animal cells, and these adhesion complexes accumulate ingrowth-limited cells adapted to saline stress.

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells (Nicotiana tabacum/gossii). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants. The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.

Synchronously dividing cells of Chlamydomonas reinhardtii Dang. (Chlorophyta) produce a single peak of cyclic adenosine monophosphate (cAMP), about sevenfold above the basal level, at the time of onset of the flagellar shortening that precedes mitosis. Cultures of a spontaneous palmelloid variant (which forms flagella-less cell clusters) of C. reinhardtii produce up to 15 times more cAMP per gram fresh weight of cells than do cultures of normal C. reinhardtii. Revertants from the palmelloid phenotype to the normal phenotype exhibit the low levels of cAMP characteristic of normal C. reinhardtii. Thus, elevation of cAMP level and decreased ability to form or maintain flagella are closely related phenomena. We propose that flagellar assembly/disassembly is regulated by endogenous cAMP in C. reinhardtii.

Cells of potato (Solanum tuberosum L.) were obtained which were capable of photoautotrophic growth in liquid suspension culture under a photon flux density of 90–110 μmol m−2 s−1 PAR and in an atmosphere enriched with 2% CO2. These photoautotrophic cells contained between 100 to 200 μg Chl (g fresh weight)−1 and fixed CO2 at a maximum rate of 16 μmol CO2 (g fresh weight)−1h−1. In order to obtain cells capable of photoautotrophic growth it was necessary to adapt highly chlorophyllous heterotrophic cells (>50 μg Chl (g fresh weight)−1) for growth in medium with 2.5 g sucrose 1−1 (photomixotrophic cells). The photomixotropic cells had a Chl content of ca 100 μg Chl (g fresh weight)−1 and were capable of photosynthetic activity which allowed them to survive after sugars had been depleted from the medium. It was from the photomixotrophic cells that cells capable of photoautotrophic growth were obtained. Heterotrophic cells initially established in liquid medium with 25 g sucrose I−1 from chlorophyllous callus contained about 50 to 150 μg Chl (g fresh weight)−1. However, after 5 to 10 passages the Chl content decreased to a maximum of 15 μg Chl (g fresh weight)−1. These cells could not be adapted to photomixotrophic or photoautotrophic growth. These cells also were not able to regain Chl or initiate high rates of CO2 fixation during the stationary phase of growth as did photomixotrophic cells or chlorophyllous heterotrophic cells. The loss of Chl exhibited by the cells during adaption to heterotrophic growth could be attributed at least in part to unbalanced growth (when cell division and growth exceeds Chl accumulation). Sucrose appeared to have an inhibitory effect directly on photosynthesis independent of Chl accumulation.