Changes of Mitochondrial Properties in Maize Seedlings Associated with Selection for Germination at Low Temperature. Fatty Acid Composition, Cytochrome c Oxidase, and Adenine Nucleotide Translocase Activities1

Mitochondria are affected by low temperature during seedling establishment in maize (Zea mays L.). We evaluated the associated changes in the mitochondrial properties of populations selected for high (C4-H) and low (C4-L) germination levels at 9.5°C. When seedlings of the two populations were grown at 14°C (near the lower growth limit), the mitochondrial inner membranes of C4-H showed a higher percentage of 18-carbon unsaturated fatty acids, a higher fluidity, and a higher activity of cytochrome c oxidase. We found a positive relationship between these properties and the activity of a mitochondrial peroxidase, allowing C4-H to reduce lipid peroxidation relative to C4-L. The specific activity of reconstituted ATP/ADP translocase was positively associated with this peroxidase activity, suggesting that translocase activity is also affected by chilling. The level of oxidative stress and defense mechanisms are differently expressed in tolerant and susceptible populations when seedlings are grown at a temperature near the lower growth limit. Thus, the interaction between membrane lipids and cytochrome c oxidase seems to play a key role in maize chilling tolerance. Furthermore, the divergent-recurrent selection procedure apparently affects the allelic frequencies of genes controlling such an interaction.

Third-generation synchrotron x-ray diffraction of 6-μm crystal of raite, ≈Na3Mn3Ti0.25Si8O20(OH)2⋅10H2O, opens up new chemistry and physics of low-temperature minerals

The crystal structure of raite was solved and refined from data collected at Beamline Insertion Device 13 at the European Synchrotron Radiation Facility, using a 3 × 3 × 65 μm single crystal. The refined lattice constants of the monoclinic unit cell are a = 15.1(1) Å; b = 17.6(1) Å; c = 5.290(4) Å; β = 100.5(2)°; space group C2/m. The structure, including all reflections, refined to a final R = 0.07. Raite occurs in hyperalkaline rocks from the Kola peninsula, Russia. The structure consists of alternating layers of a hexagonal chicken-wire pattern of 6-membered SiO4 rings. Tetrahedral apices of a chain of Si six-rings, parallel to the c-axis, alternate in pointing up and down. Two six-ring Si layers are connected by edge-sharing octahedral bands of Na+ and Mn3+ also parallel to c. The band consists of the alternation of finite Mn–Mn and Na–Mn–Na chains. As a consequence of the misfit between octahedral and tetrahedral elements, regions of the Si–O layers are arched and form one-dimensional channels bounded by 12 Si tetrahedra and 2 Na octahedra. The channels along the short c-axis in raite are filled by isolated Na(OH,H2O)6 octahedra. The distorted octahedrally coordinated Ti4+ also resides in the channel and provides the weak linkage of these isolated Na octahedra and the mixed octahedral tetrahedral framework. Raite is structurally related to intersilite, palygorskite, sepiolite, and amphibole.

Nuclear Activities of Basic Fibroblast Growth Factor: Potentiation of Low-Serum Growth Mediated by Natural or Chimeric Nuclear Localization Signals

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways.

The Arabidopsis CBF Gene Family Is Composed of Three Genes Encoding AP2 Domain-Containing Proteins Whose Expression Is Regulated by Low Temperature but Not by Abscisic Acid or Dehydration1

We have identified two genes from Arabidopsis that show high similarity with CBF1, a gene encoding an AP2 domain-containing transcriptional activator that binds to the low-temperature-responsive element CCGAC and induces the expression of some cold-regulated genes, increasing plant freezing tolerance. These two genes, which we have named CBF2 and CBF3, also encode proteins containing AP2 DNA-binding motifs. Furthermore, like CBF1, CBF2 and CBF3 proteins also include putative nuclear-localization signals and potential acidic activation domains. The CBF2 and CBF3 genes are linked to CBF1, constituting a cluster on the bottom arm of chromosome IV. The high level of similarity among the three CBF genes, their tandem organization, and the fact that they have the same transcriptional orientation all suggest a common origin. CBF1, CBF2, and CBF3 show identical expression patterns, being induced very rapidly by low-temperature treatment. However, in contrast to most of the cold-induced plant genes characterized, they are not responsive to abscisic acid or dehydration. Taken together, all of these data suggest that CBF2 and CBF3 may function as transcriptional activators, controlling the level of low-temperature gene expression and promoting freezing tolerance through an abscisic acid-independent pathway.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Mimicry of the calcium-induced conformational state of troponin C by low temperature under pressure.

Calcium binding to the N-domain of troponin C initiates a series of conformational changes that lead to muscle contraction. Calcium binding provides the free energy for a hydrophobic region in the core of N-domain to assume a more open configuration. Fluorescence measurements on a tryptophan mutant (F29W) show that a similar conformational change occurs in the absence of Ca2+ when the temperature is lowered under pressure. The conformation induced by subzero temperatures binds the hydrophobic probe bis-aminonaphthalene sulfonate, and the tryptophan has the same fluorescence lifetime (7 ns) as in the Ca2+-bound form. The decrease in volume (delta V = -25.4 ml/mol) corresponds to an increase in surface area. Thermodynamic measurements suggest an enthalpy-driven conformational change that leads to an intermediate with an exposed N-domain core and a high affinity for Ca2+.

Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants.

Using tobacco plants that had been transformed with the cDNA for glycerol-3-phosphate acyltransferase, we have demonstrated that chilling tolerance is affected by the levels of unsaturated membrane lipids. In the present study, we examined the effects of the transformation of tobacco plants with cDNA for glycerol-3-phosphate acyltransferase from squash on the unsaturation of fatty acids in thylakoid membrane lipids and the response of photosynthesis to various temperatures. Of the four major lipid classes isolated from the thylakoid membranes, phosphatidylglycerol showed the most conspicuous decrease in the level of unsaturation in the transformed plants. The isolated thylakoid membranes from wild-type and transgenic plants did not significantly differ from each other in terms of the sensitivity of photosystem II to high and low temperatures and also to photoinhibition. However, leaves of the transformed plants were more sensitive to photoinhibition than those of wild-type plants. Moreover, the recovery of photosynthesis from photoinhibition in leaves of wild-type plants was faster than that in leaves of the transgenic tobacco plants. These results suggest that unsaturation of fatty acids of phosphatidylglycerol in thylakoid membranes stabilizes the photosynthetic machinery against low-temperature photoinhibition by accelerating the recovery of the photosystem II protein complex.

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature1

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m−2 s−1) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m−2 s−1) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.

Separating Growth from Elastic Deformation during Cell Enlargement1

Plants change size by deforming reversibly (elastically) whenever turgor pressure changes, and by growing. The elastic deformation is independent of growth because it occurs in nongrowing cells. Its occurrence with growth has prevented growth from being observed alone. We investigated whether the two processes could be separated in internode cells of Chara corallina Klien ex Willd., em R.D.W. by injecting or removing cell solution with a pressure probe to change turgor while the cell length was continuously measured. Cell size changed immediately when turgor changed, and growth rates appeared to be altered. Low temperature eliminated growth but did not alter the elastic effects. This allowed elastic deformation measured at low temperature to be subtracted from elongation at warm temperature in the same cell. After the subtraction, growth alone could be observed for the first time. Alterations in turgor caused growth to change rapidly to a new, steady rate with no evidence of rapid adjustments in wall properties. This turgor response, together with the marked sensitivity of growth to temperature, suggested that the growth rate was not controlled by inert polymer extension but rather by biochemical reactions that include a turgor-sensitive step.

Interaction of Osmotic Stress, Temperature, and Abscisic Acid in the Regulation of Gene Expression in Arabidopsis

The impact of simultaneous environmental stresses on plants and how they respond to combined stresses compared with single stresses is largely unclear. By using a transgene (RD29A-LUC) consisting of the firefly luciferase coding sequence (LUC) driven by the stress-responsive RD29A promoter, we investigated the interactive effects of temperature, osmotic stress, and the phytohormone abscisic acid (ABA) in the regulation of gene expression in Arabidopsis seedlings. Results indicated that both positive and negative interactions exist among the studied stress factors in regulating gene expression. At a normal growth temperature (22°C), osmotic stress and ABA act synergistically to induce the transgene expression. Low temperature inhibits the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas low temperature and ABA treatments are additive in inducing transgene expression. Although high temperature alone does not activate the transgene, it significantly amplifies the effects of ABA and osmotic stress. The effect of multiple stresses in the regulation of RD29A-LUC expression in signal transduction mutants was also studied. The results are discussed in the context of cold and osmotic stress signal transduction pathways.