Arabidopsis Sucrose Transporter SUT4 Interacts with Cytochrome b5-2 to Regulate Seed Germination in Response to Sucrose and Glucose

It remains unknown whether a sucrose transporter mediates sugar signaling. Here, we report that the Arabidopsis （Arabidopsis thaliana） sucrose transporter SUT4 interacts with five members of the Arabidopsis cytochrome b5 （Cyb5） family, and sucrose represses the interaction between SUT4 and a Cyb5 member Cyb5-2/A. We observed that down- regulation of SUT4 and three cytochrome b5 members （Cyb5-2, Cyb5-4, and Cyb5-6） confers the sucrose- and glucose- insensitive phenotypes in the sucrose/glucose-induced inhibition of seed germination. The sut4 cybS-2 double mutant displays slightly stronger sucrose/glucose-insensitive phenotypes than either the sut4 or cyb5-2 single mutant. We showed that the SUT4/Cyb5-2-mediated signaling in the sucrose/glucose-induced inhibition of seed germination does not require ABA or the currently known ABI2/ABI4/ABI5-mediated signaling pathway（s）. These data provide evidence that the sucrose transporter SUT4 interacts with Cyb5 to positively mediate sucrose and glucose signaling in the sucrose/ glucose-induced inhibition of seed germination.

Cytochrome b5 Reductase 1 Triggers Serial Reactions that Lead to Iron Uptake in Plants

Rhizosphere acidification is essential for iron （Fe） uptake into plant roots. Plasma membrane （PM） H＊-ATPases play key roles in rhizosphere acidification. However, it is not fully understood how PM H＋-ATPase activity is regulated to enhance root Fe uptake under Fe-deficient conditions. Here, we present evidence that cytochrome b5 reductase 1 （CBR1） increases the levels of unsaturated fatty acids, which stimulate PM H＋-ATPase activity and thus lead to rhizosphere acidification. CBRl-overexpressing （CBRI-OX） Arabidopsis thaliana plants had higher levels of unsaturated fatty acids （18：2 and 18：3）, higher PM H＊-ATPase activity, and lower rhizosphere pH than wild-type plants. By contrast, cbrl loss-of-function mutant plants showed lower levels of unsaturated fatty acids and lower PM H＊-ATPase activity but higher rhizosphere pH. Reduced PM H＊-ATPase activity in cbrl could be restored in vitro by addition of unsatu- rated fatty acids. Transcript levels of CBR1, fatty acids desaturase 2 （FAD2）, and fatty acids desaturase 3 （FAD3） were increased under Fe-deficient conditions. We propose that CBR1 has a crucial role in increasing the levels of unsaturated fatty acids, which activate the PM H＊-ATPase and thus reduce rhizosphere pH. This reaction cascade ultimately promotes root Fe uptake.

4-Hydroxycinnamic acid attenuates neuronal cell death by inducing expression of plasma membrane redox enzymes and improving mitochondrial functions

Many approaches to neurodegenerative diseases that focus on amyloid-βclearance and gene therapy have not been successful.Some therapeutic applications focus on enhancing neuronal cell survival during the pathogenesis of neurodegenerative diseases,including mitochondrial dysfunction.Plasma membrane(PM)redox enzymes are crucial in maintaining cellular physiology and redox homeostasis in response to mitochondrial dysfunction.Neurohormetic phytochemicals are known to induce the expression of detoxifying enzymes under stress conditions.In this study,mechanisms of neuroprotective effects of 4-hydroxycinnamic acid(HCA)were examined by analyzing cell survival,levels of abnormal proteins,and mitochondrial functions in two different neuronal cells.HCA protected two neuronal cells exhibited high expression of PM redox enzymes and the consequent increase in the NAD^(+)/NADH ratio.Cells cultured with HCA showed delayed apoptosis and decreased oxidative/nitrative damage accompanied by decreased ROS production in the mitochondria.HCA increased the mitochondrial complexes I and II activities and ATP production.Also,HCA increased mitochondrial fusion and decreased mitochondrial fission.Overall,HCA maintains redox homeostasis and energy metabolism under oxidative/metabolic stress conditions.These findings suggest that HCA could be a promising therapeutic approach for neurodegenerative diseases.

New insights into the role of cytochrome P450 reductase (POR) in microsomal redox biology

Cytochrome P450 reductase(POR)is an essential electron transfer protein located on the endoplasmic reticulum of most cell types,and has long been appreciated for its role in cytochrome P450-mediated drug metabolism.Additional roles and electron acceptors for POR have been described,but it is largely with the recent availability of POR-null tissues that these supplemental roles for POR have been able to be explored.These studies have confirmed POR as the principal redox partner for the microsomal P450s responsible for drug and xenobiotic metabolism as well as cholesterol and bile acid synthesis,and for heme oxygenase,which catalyzes the initial step in the breakdown of heme.Surprisingly,these studies have revealed that squalene monooxygenase,an enzyme essential to cholesterol synthesis,has a second unknown redox partner in addition to POR,and that 7-dehydrocholesterol reductase,previously proposed to require POR as an electron donor,functions fully independently of POR.These studies have also helped define the role of cytochrome b5 in P450 catalysis,and raise the question as to the extent to which POR contributes to b5-dependent redox pathways.

Influences of the Hydrophobicity of the Heme-binding Pocket on the Properties and Functions of Cytochrome b_5 Mutants

The mutation sites of the four mutants F35Y, P40V, V45E and V45Y of cytochrome b 5 are located at the edge of the heme binding pocket. The solvent accessible areas of the “pocket interior” of the four mutants and the wild type cytochrome b 5 have been calculated based on their crystal structures at high resolution. The change in the hydrophobicity of the heme binding pocket resulting from the mutation can be quantitatively described using the difference of the solvent accessible area of the “pocket interior” of each mutant from that of the wild type cytochrome b 5. The influences of the hydrophobicity of the heme binding pocket on the protein stability and redox potential are discussed.

Structures of Cytochrome b_5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c

Glu44, Glu48, Glu56 and Asp60 are the negatively charged residues located at the molecular surface of cytochrome b 5. Two mutants of cytochrome b 5 were prepared, in which two or all of these four residues were mutated to alanines. The mutations give rise to slightly positive shifts of the redox potentials of cytochrome b 5 and obvious decrease of the cytochrome b 5-cytochrome c binding constants and electron transfer rates. The crystal structures of the two mutants were determined at 0.18 nm resolution, showing no alteration in overall structures and exhibiting slight changes in the local conformations around the mutation sites as compared with the wild-type protein. Based on the crystal structure of the quadruple-site mutant, a model for the binding of this mutant with cytochrome c is proposed, which involves the salt bridges from Glu37, Glu38 and heme propionate of cytochrome b 5 to three lysines of cytochrome c and can well account for the properties and behaviors of this mutant.

Proline-40 is Essential to Maintaining Cytochrome b_5's Stability and Its Electron Transfer with Cytochrome c

In order to illustrate the roles played by Pro40 in the structure, properties and functions of Cytochrome b 5, three mutated genes, P40V, P40Y, P40G were constructed in this work. Only the P40V gene was successfully expressed into holoprotein in E. coli JM83. According to the results of X-ray crystallographic analysis and various kinds of spectroscopy studies, it is evident that substituting valine for Pro40 does not result in significant alterations in the protein's overall structure; however, local conformational perturbations in the proximity of the heme do occur. The redox potential of the P40V mutant is 40 mV lower than that of the wild type protein. Its stability towards heat, urea, acid and ethanol were significantly decreased. The mutation leads to a decrease in the hydrophobicity of the heme pocket, which is probably the major factor contributing to the above changes. Binding constants and electron transfer rates between cytochrome b 5 and cytochrome c were determined using UV-visible spectroscopy and stopped-flow techniques for both the wild type and the mutant. The results showed that the substitution of Pro40 by valine does not influence the binding constant of cytochrome b 5 to cytochrome c; however, the electron transfer rate between them decreased significantly. This indicates that proline-40 is essential to maintaining cytochrome b 5's stability and its electron transfer with cytochrome c. These studies also provided a good example that property and functional changes of a protein do not necessarily require large overall structural alterations; in most cases, only perturbations on the local conformations are sufficient to induce significant changes in protein′s properties and functions.