Thus, characterization of FBNs function in rice would increase our knowledge about maintaining photosynthetic efficiency and stress tolerance. These results provide strong evidence that the function of FBN5 in PQ-9 biosynthesis is well conserved between eudicots and monocots. Rice seeds were germinated on 0. Total lipids were extracted from frozen tissue grindates in liquid nitrogen in cold ethyl acetate.
After centrifugation, the supernatant was transferred to a new tube and evaporated under nitrogen. PQ-9 was detected by absorption at nm, while PC-8 and tocopherols were detected fluorimetrically nm excitation and nm emission. The compounds were quantified by comparison to their corresponding external calibration standards, and the data were corrected by comparison with the recovery of rac-Tocol Matreya, PA, United States as the internal standard.
For the analysis of carotenoids and chlorophylls, the chromatographic conditions were the same as above. The HPLC peak areas at nm were integrated. The vectors were delivered into maize mesophyll protoplasts using the polyethylene glycol-calcium mediated method, followed by 12—16 h incubation to allow transient expression Cho et al. Chlorophyll autofluorescence was used as a chloroplast marker.
OsFBN5 expression was detected in the complemented fbn plants using the appropriate primer pairs Supplementary Table 1. Actin2 was used as an internal control. The protein sequences of the individual FBN family members from Arabidopsis were used as queries against the rice genome.
Sequence alignment was performed using the default ClustalW parameters and maximum likelihood was used for tree construction in MEGA6 program. To investigate the roles of FBNs in rice, a monocot model species, a phylogenetic tree was constructed based on amino acid sequences of FBNs from Arabidopsis and rice. Analysis of putative physicochemical properties, isoelectric points PIs , and hydrophobicities of the Arabidopsis and rice FBN sequences after removal of the predicted plastid targeting peptides revealed predicted similar physicochemical properties between the Arabidopsis and rice FBN homologs Supplementary Figure 2.
The product of this transcript was altered in 14 amino acids, including a 10 amino acid deletion at the C-terminus, indicating that these residues are indispensable for AtFBN5 function.
Plastid targeting peptides green box were predicted by the ChloroP program. Asterisks indicate identical residues between Arabidopsis and rice. Unlike the Atfbn plants, the Osfbn5 homozygous mutant plants were not seedling lethal. When the WT and Osfbn5 homozygous mutant plants were transplanted onto soil in the greenhouse, the mutant plants rapidly dried and eventually died, while the WT plants grew and yielded seeds.
Isolation and growth phenotypes of the OsFBN5 mutant alleles. Black boxes and black lines represent exons and introns, respectively. The Tos17 insertion sites for two independent mutant lines, Osfbn and Osfbn, are indicated by red lines.
VTE5 has been characterized in Arabidopsis where its mutant allele, vte5, causes a substantial reduction of the tocopherol content in seeds and to a lesser extent in leaves Valentin et al.
Furthermore, based on sequence similarity, a locus encoding a putative VTE5 paralog was identified in Arabidopsis, which further was characterized as a farnesol kinase FOLK Fitzpatrick et al. However, its involvement in tocopherol biosynthesis was not addressed.
So far, VTE5 has only been characterized in Arabidopsis, and its contribution to tocopherol content is largely unknown in other species and organs, such as in edible fleshy fruits.
Moreover, the impact of VTE5 deficiency on plant metabolism remains unexplored. Solanum lycopersicum is an interesting model species for studying tocopherol metabolism. Besides being an important food crop worldwide, the fruits are a significant source of VTE for the human diet Chun et al.
Additionally, tomato ripening, which encompasses the conversion of chloroplasts into chromoplasts, couples Chl degradation and an active MEP pathway Seymour et al. A previous study on the regulation of tocopherol biosynthesis in this species demonstrated a strong correlation between VTE5 mRNA levels and the contents of Chl and tocopherol in tomato leaves and fruits, suggesting the contribution of phytol recycling to tocopherol biosynthesis Quadrana et al.
Moreover, expression analysis of senescence-related tomato mutants suggested that maintenance of the de novo phytyl diphosphate synthesis might, at later ripening stages, compensate for the lack of Chl-derived phytol for tocopherol production in fruits Almeida et al. All parameters used in the program were at the default settings. Because S. The UbiA-1 protein from S. Indeed, the result of an orthologue analysis using the www server of the Microbial Genome Data Base mbgd. On the other hand, UbiA-2 was treated in the data base as an orthologue of cyanobacterial chlorophyll a synthase ChlG, which is also homologous with UbiA prenyltransferase and catalyzes the transfer of a phytyl group to chlorophyllide a.
Moreover, almost all archaea whose entire genome sequences have been determined, except for Nanoarchaeum equitans, appeared to possess the predicted orthologues of UbiA Because it is generally thought that archaea do not require either chlorophyll or bacteriochlorophyll, we initially thought that UbiA-2 might be involved in the biosynthesis of prenylated hemes, which have the porphyrin structure of chlorophyll.
However, we immediately noted that the genome of S. These facts strongly suggest that UbiA-2 and its predicted orthologues encoded in the archaeal genomes have an unrevealed, but very important function in archaeal cells. The archaeal lipid-biosynthetic reaction in which a geranylgeranyl group is transformed to GGGP, an acyclic prenyl-acceptor, to produce DGGGP was a promising candidate for the function, although the other reactions catalyzed by the homologues of UbiA studied thus far typically require acceptors that contain aromatic or porphyrin structures.
Figure 4 Recovery of the ground state absorption bleaching upon saturating excitation of WSCP Chl a in oxic red and anoxic black conditions, with the respective fitting curves. Low intensity probing light beam centered at the Soret band. Full size image Schmidt et al.
This would imply that the shortening of the lifetime of 3Chl that we observed in the presence of O2 should take place without generating the oxidative species 1O2. In order to verify this possibility, we measured the photo-induced 1O2 yield in our system. This sensor selectively reacts with 1O2, leading to an endo-peroxidized product with a highly increased fluorescence quantum yield. The measurements of 1O2 were accompanied by photobleaching measurements, performed on the same samples, by means of Chl fluorescence detection.
The two Chl-binding complexes display comparable photobleaching, largely reduced in comparison to a reference sample containing free Chl a in octyl glucoside OG micelles. Emission before illumination: F0. Background signals were subtracted. Full size image Surprisingly and in marked contrast to the previous work of Schmidt et al.
A reference sample with unbound Chl a in OG solution displayed, under the same experimental conditions, an unexpectedly low 1O2 evolution. However, the reason for this behavior can be attributed to the concomitant high degree of photobleaching: due to the fast reaction with 1O2, the number of excitable Chls quickly decreases, and consequently less and less 1O2 is photosensitized at increasing time intervals.
When working in the same experimental conditions described by Schmidt et al. S2A , we also measured a low 1O2 yield. The different quantification of 1O2 measured by SOSG and spin probe methods may be related to the different repartition coefficients of the probes between the micellar and the aqueous phases.
S2A , confirming that TEMP is sequestered away from the aqueous phase in the presence of the micelles. Therefore the underestimate of the 1O2 photosensitization in the previous report 5 is due to the interfering presence of DM micelles. The decrease of the signal under this condition can be attributed to the fact that in homogeneous solution the increase in the local concentration, generated in micelles by the confinement of Chls and TEMP, is lost, and the probability of encounter between TEMP and 1O2 is consequently diminished.
In conclusion, our experimental approach based on two different methods for determining the 1O2 production led to the conclusion that 1O2 production in WSCP is not suppressed but is of the same order as that of free Chl and larger than that observed in LHCII Fig.
How plants co-ordinate isoprenoid precursor distribution for supplying biosynthesis of tocopherol and other prenyllipids in different organs is poorly understood. Similar to Arabidopsis Block et al. Moreover, almost all archaea whose entire genome sequences have been determined, except for Nanoarchaeum equitans, appeared to possess the predicted orthologues of UbiA In addition to the absorption peak of GGGP, which eluted at about 27 min, a peak eluting at about 35 min was observed. In order to get insight into the mechanism responsible for the photostability of WSCP, we performed a systematic study using electron paramagnetic resonance EPR techniques to determine the properties of the photoexcited 3Chl a, flash photolysis to measure the 3Chl a lifetime, and trapping experiments to evaluate the 1O2 yield.
In this study, we identified a gene encoding a homologue of ubiquinone-biosynthetic prenyltransferase from the whole genome sequence of a thermoacidophilic archaeon Sulfolobus solfataricus as the candidate of the DGGGPS gene. Photoinactivation of Osfbn5 homozygous mutant plants was light intensity dependent Figure 3. Figure 4 Recovery of the ground state absorption bleaching upon saturating excitation of WSCP Chl a in oxic red and anoxic black conditions, with the respective fitting curves. Moreover, the lifetime of the 3Chl generated in WSCPs is comparable to that observed in other Chl-containing systems and is reduced in presence of oxygen.
In contrast to previous observations, we found that WSCP actually photosensitizes singlet oxygen with an efficiency comparable to that of Chl in organic solvent. The small hfcs, which are well resolved in our Mims spectra, were neither assigned nor detected in previous pulse ENDOR studies, and will require a further computational analysis to reach a reliable and complete assignment. Alternatively, Boeux-Fontvieille et al. Figure 4 Recovery of the ground state absorption bleaching upon saturating excitation of WSCP Chl a in oxic red and anoxic black conditions, with the respective fitting curves.
For the analysis of carotenoids and chlorophylls, the chromatographic conditions were the same as above.