Different Cis-Regulatory Elements Control the Tissue-Specific Contribution of Plastid ω-3 Desaturases to Wounding and Hormone Responses.

Trienoic fatty acids are essential constituents of biomembranes and precursors of jasmonates involved in plant defense responses. Two ω-3 desaturases, AtFAD7 and AtFAD8, synthetize trienoic fatty acids in the plastid. Promoter:GUS and mutagenesis analysis was used to identify cis-elements controlling AtFAD7 and AtFAD8 basal expression and their response to hormones or wounding. AtFAD7 promoter GUS activity was much higher than that of AtFAD8 in leaves, with specific AtFAD7 expression in the flower stamen and pistil and root meristem and vasculature. This specific tissue and organ expression of AtFAD7 was controlled by different cis-elements. Thus, promoter deletion and mutagenesis analysis indicated that WRKY proteins might be essential for basal expression of AtFAD7 in leaves. Two MYB target sequences present in the AtFAD7 promoter might be responsible for its expression in the flower stamen and stigma of the pistil and in the root meristem, and for the AtFAD7 wound-specific response. Two MYB target sequences detected in the distal region of the AtFAD8 gene promoter seemed to negatively control AtFAD8 expression, particularly in true leaves and flowers, suggesting that MYB transcription factors act as repressors of AtFAD8 gene basal expression, modulating the different relative abundance of both plastid ω-3 desaturases at the transcriptional level. Our data showed that the two ABA repression sequences detected in the AtFAD7 promoter were functional, suggesting an ABA-dependent mechanism involved in the different regulation of both ω-3 plastid desaturases. These results reveal the implication of different signaling pathways for the concerted regulation of trienoic fatty acid content in Arabidopsis.

Effects of Chlorophyll Triplet States on the Kinetics of Spectral Hole Growth.

Spectral hole burning has been employed for decades to study various amorphous solids and proteins. Triplet states and respective transient holes were incorporated into theoretical models and software simulating nonphotochemical spectral hole burning (NPHB) and including all relevant distributions, in particular the distribution of the angle between the electric field of light E and transient dipole moment of the chromophore µ. The presence of a chlorophyll a triplet state with a lifetime of several milliseconds explains the slowdown of NPHB (on the depth vs illumination dose scale) with the increase of the light intensity, as well as larger hole depths observed in weak probe beam experiments, compared to those deduced from the hole growth kinetics (HGK) measurements (signal collected at a fixed wavelength while a stronger burning beam is on) in cytochrome b6f and chemically modified LH2. We also considered the solvent deuteration effects on triplet lifetime and concluded that both triplet states and local heating likely play a role in slowing down the HGK with increasing burn intensity.

Functional analysis of ß-ketoacyl-CoA synthase from biofuel feedstock Thlaspi arvense reveals differences in the triacylglycerol biosynthetic pathway among Brassicaceae.

KEY MESSAGE: Differences in FAE1 enzyme affinity for the acyl-CoA substrates, as well as the balance between the different pathways involved in their incorporation to triacylglycerol might be determinant of the different composition of the seed oil in Brassicaceae. Brassicaceae present a great heterogeneity of seed oil and fatty acid composition, accumulating Very Long Chain Fatty Acids with industrial applications. However, the molecular determinants of these differences remain elusive. We have studied the ß-ketoacyl-CoA synthase from the high erucic feedstock Thlaspi arvense (Pennycress). Functional characterization of the Pennycress FAE1 enzyme was performed in two Arabidopsis backgrounds; Col-0, with less than 2.5% of erucic acid in its seed oil and the fae1-1 mutant, deficient in FAE1 activity, that did not accumulate erucic acid. Seed-specific expression of the Pennycress FAE1 gene in Col-0 resulted in a 3 to fourfold increase of erucic acid content in the seed oil. This increase was concomitant with a decrease of eicosenoic acid levels without changes in oleic ones. Interestingly, only small changes in eicosenoic and erucic acid levels occurred when the Pennycress FAE1 gene was expressed in the fae1-1 mutant, with high levels of oleic acid available for elongation, suggesting that the Pennycress FAE1 enzyme showed higher affinity for eicosenoic acid substrates, than for oleic ones in Arabidopsis. Erucic acid was incorporated to triacylglycerol in the transgenic lines without significant changes in their levels in the diacylglycerol fraction, suggesting that erucic acid was preferentially incorporated to triacylglycerol via DGAT1. Expression analysis of FAE1, AtDGAT1, AtLPCAT1 and AtPDAT1 genes in the transgenic lines further supported this conclusion. Differences in FAE1 affinity for the oleic and eicosenoic substrates among Brassicaceae, as well as their incorporation to triacylglycerol might explain the differences in composition of their seed oil.

Evidence of Simultaneous Spectral Hole Burning Involving Two Tiers of the Protein Energy Landscape in Cytochrome b6f.

Cytochrome b6f, with one chlorophyll molecule per protein monomer, is a simple model system whose studies can help achieve a better understanding of nonphotochemical spectral hole burning (NPHB) and single-complex spectroscopy results obtained in more complicated photosynthetic chlorophyll-protein complexes. We are reporting new data and proposing an alternative explanation for spectral dynamics that was recently observed in cytochrome b6f using NPHB. The relevant distribution of the tunneling parameter λ is a superposition of two components that are nearly degenerate in terms of the resultant NPHB yield and represent two tiers of the energy landscape responsible for NPHB. These two components likely burn competitively; we present the first demonstration of modeling a competitive NPHB process. Similar values of the NPHB yield result from distinctly different combinations of barrier heights, shifts along the generalized coordinate d, and/or masses of the entities involved in conformational changes m, with md2 parameter different by a factor of 2.7. Consequently, in cytochrome b6f, the first (at least) 10 h of fixed-temperature recovery preferentially probe different components of the barrier- and λ-distributions encoded into the spectral holes than thermocycling experiments. Both components most likely represent dynamics of the protein and not of the surrounding buffer/glycerol glass.

Probing Energy Landscapes of Cytochrome b6f with Spectral Hole Burning: Effects of Deuterated Solvent and Detergent.

In non-photochemical spectral hole burning (NPHB) and spectral hole recovery experiments, cytochrome b6f protein exhibits behavior that is almost independent of the deuteration of the buffer/glycerol glassy matrix containing the protein, apart from some differences in heat dissipation. On the other hand, strong dependence of the hole burning properties on sample preparation procedures was observed and attributed to a large increase of the electron-phonon coupling and shortening of the excited-state lifetime occurring when n-dodecyl ß-d-maltoside (DM) is used as a detergent instead of n-octyl ß-d-glucopyranoside (OGP). The data was analyzed assuming that the tunneling parameter distribution or barrier distribution probed by NPHB and encoded into the spectral holes contains contributions from two nonidentical components with accidentally degenerate excited state λ-distributions. Both components likely reflect protein dynamics, although with some small probability one of them (with larger md2) may still represent the dynamics involving specifically the -OH groups of the water/glycerol solvent. Single proton tunneling in the water/glycerol solvent environment or in the protein can be safely excluded as the origin of observed NPHB and hole recovery dynamics. The intensity dependence of the hole growth kinetics in deuterated samples likely reflects differences in heat dissipation between protonated and deuterated samples. These differences are most probably due to the higher interface thermal resistivity between (still protonated) protein and deuterated water/glycerol outside environment.

A simple and efficient method to prepare pure dimers and monomers of the cytochrome b 6 f complex from spinach.

Using a single size-exclusion chromatography we were able to isolate highly pure dimers and monomers of the Cyt b 6 f complex from spinach from a bulk preparation of that protein complex obtained with a standard procedure. At higher protein/detergent ratio during the chromatography most of the Cyt b 6 f complex remained as dimers. In contrast, at lower protein/detergent ratio (around 15 times lower), most dimers became monomerized. As a bonus, this chromatography also allowed the elimination of potential Chl a contaminant to the Cyt b 6 f preparations. SDS-PAGE protein analysis with 18% (w/v) acrylamide revealed the loss of the ISP subunit in our monomeric preparation. However, it fully retained the content of Chl a, a prerequisite to perform any spectroscopic study involving this unique pigment.

In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.

The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and ß. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the ß-subunit (ß/ß) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane.

Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b(559) from higher plants.

A study of the in vitro reconstitution of sugar beet cytochrome b(559) of the photosystem II is described. Both α and ß cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ßß) homodimers and (αß) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b(559) form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b(559). Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b(559) subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the ß subunit from the cyanobacterium the formation of a cytochrome b(559)-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ßß) homodimer cytochrome b(559)-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b(559). A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.

Characterization of the recombinant copper chaperone (CCS) from the plant Glycine (G.) max.

The goal of the present work was to characterize the recombinant copper chaperone (CCS) from soybean. Very little is known about plant copper chaperones, which makes this study of current interest, and allows for a comparison with the better known homologues from yeast and humans. To obtain sizeable amounts of pure protein suitable for spectroscopic characterization, we cloned and overexpressed the G. max CCS chaperone in E. coli in the presence of 0.5 mM CuSO(4) and 0.5 mM ZnSO(4) in the broth. A pure protein preparation was obtained by using two IMAC steps and pH gradient chromatography. Most of the proteins were obtained as apo-form, devoid of copper atoms. The chaperone showed a high content (i.e., over 40%) of loops, turns and random coil as determined both by circular dichroism and homology modelling. The homology 3-D structural model suggests the protein might fold in three structural protein domains. The 3-D model along with the primary structure and spectroscopic data may suggest that copper atoms occupy the two metal binding sites, MKCEGC and CTC, within the N-terminal domain I and C-terminal domain III, respectively. But only one Zn-binding site was obtained spectroscopically.