SummaryGlycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCI, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCI, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCI for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.
Reaction centers of wild-type Rhodobacter sphaeroides were selectively (13)C-isotope labeled in bacteriochlorophyll and bacteriopheophytin. (13)C solid-state CP/MAS NMR and photo-CIDNP were used to provide insight into the electronic structure of the primary electron donor and acceptor on the atomic scale. The first 2-dimensional photochemically induced dynamic nuclear polarization (photo-CIDNP) (13)C-(13)C solid-state MAS NMR spectra reveal that negative charging of the two BChl rings of the primary donor is involved in ground-state tuning of the oxidation potential of these cofactors in the protein via local electrostatic interactions. In particular, the (13)C shifts show moderate differences in the electronic structure between the two BChl molecules of the special pair in the electronic ground state, which can be attributed to hydrogen bonding of one of the BChl molecules. The major fraction of the electron spin density is strongly delocalized over the two BChl molecules of the special pair and the photochemically active BPhe. A small fraction of the pi-spin density is distributed over a fourth component, which is assigned to the accessory BChl. Comparison of the photo-CIDNP data with "dark" NMR spectra obtained in ultra high field indicates a rigid special pair environment upon photoreaction and suggests that structural changes of the aromatic macrocycles of the two BChl molecules of the special pair do not significantly contribute to the reorganization energy associated with the charge-transfer process.
Photochemically induced dynamic nuclear polarization (photo-CIDNP) is observed in photosynthetic reaction centers of the carotenoid-less strain R26 of the purple bacterium Rhodobacter sphaeroides by (13)C solid-state NMR at three different magnetic fields (4.7, 9.4, and 17.6 T). The signals of the donor appear enhanced absorptive (positive) and of the acceptor emissive (negative). This spectral feature is in contrast to photo-CIDNP data of reactions centers of Rhodobacter sphaeroides wildtype reported previously (Prakash, S.; Alia; Gast, P.; de Groot, H. J. M.; Jeschke, G.; Matysik, J. J. Am. Chem. Soc. 2005, 127, 14290-14298) in which all signals appear emissive. The difference is due to an additional mechanism occurring in RCs of R26 in the long-living triplet state of the donor, allowing for spectral editing by different enhancement mechanisms. The overall shape of the spectra remains independent of the magnetic field. The strongest enhancement is observed at 4.7 T, enabling the observation of photo-CIDNP enhanced NMR signals from reaction center cofactors in entire bacterial cells allowing for detection of subtle changes in the electronic structure at nanomolar concentration of the donor cofactor. Therefore, we establish in this paper photo-CIDNP MAS NMR as a method to study the electronic structure of photosynthetic cofactors at the molecular and atomic resolution as well as at cellular concentrations.
We report 13 C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo-CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13 C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680 .؉ . Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P 680 . ؉ . PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.
SummaryArabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis, which encodes choline oxidase, the enzyme that synthesizes glycinebetaine from choline. The transformation enabled the plants to accumulate glycinebetaine in chloroplasts, and signi®cantly enhanced the freezing tolerance of plants. Furthermore, the photosynthetic machinery of transformed plants was more tolerant to freezing stress than that of wild-type plants. Exogenous application of glycinebetaine also increased the freezing tolerance of wild-type plants, suggesting that the presence of glycinebetaine in transformed plants had enhanced their ability to tolerate freezing stress. Northern blotting analysis revealed that the enhancement of freezing tolerance was not related to the expression of four cold-regulated genes. These results suggest that engineering of the biosynthesis of glycinebetaine by transformation with the codA gene might be an effective method for enhancing the freezing tolerance of plants.
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