Kuang Tingyun

The characteristics of CO2 assimilation of photosynthesis and chlorophyll fluorescence in transgenic PEPC rice

With PEPC, PPDK, NADP-ME and PEPC+ PPDK transgenic and untransformed rice (Orysa sativa L.), the activities of related C4 photosynthesis enzymes, the chlorophyll fluorescence parameters, CO2 exchange and other physiological indexes were compared, in which the physiological characteristics of PEPC transgenic rice were mainly studied. The results were as follows: ( i ) The activities of PEPC in PEPC transgenic rice were 20-fold higher than those in untransformed rice; the light-saturation photosynthetic rates and the carboxylation efficiency of PEPC transgenic rice were increased by 55% and 50% more than those of untransformed rice, respectively, while the CO2 compensation point decreased by 27%. (ii) The PS II photochemical efficiency (Fv/Fm) and photochemical quenching (qp) of transgenic PEPC rice decreased less in comparison with those of untransformed rice after the treatment with high light intensity (3 h) or methyl viologen (MV), a photooxidative reagent, which demonstrated that the tolerance of PEPC transgenic rice to photoinhibition and photooxidation was enhanced. (iii) Under the condition of high light intensity, the activity of RuBPCase in PEPC transgenic rice did not obviously vary while the activity induced of carbonic anhydrase (CA) in PEPC transgenic rice increased by 1.8 fold. These results would provide some beneficial enlightment for revealing the mechanism of high photosynthetic efficiency and breeding with high photosynthetic efficiency in rice.

EfficientAgrobacterium-mediated transformation of rice by phosphomannose isomerase/mannose selection

A positive selection system was developed forAgrobacterium-mediated transformation of rice that does not use toxic compounds such as antibiotics or herbicides. The selection system is based on theEscherichia coli phosphomannose isomerase (pmi) gene as a selectable marker and mannose as the selective agent. Only transgenic plants were able to metabolize mannose into a usable source of carbon, fructose. Selection was achieved using a combination of mannose and sucrose at 10 g/L and 5 g/L, respectively. Transgenic rice plants were produced efficiently injapanica rice variety Zhonghua 8, with transformation frequency of 16.5%, which was slightly lower than that achieved by hygromycin selection.

Investigation of Chlorophyll Protein 43 and 47 Denaturation by Terahertz Time-Domain Spectroscopy

We demonstrate the applications of terahertz time-domain spectroscopy to distinguish conformation changes of the chlorophyll proteins CP43 and CP47 induced by the treatment of guanidine hydrochloride, light irradiation and heating. It is indicated that THz transmission spectroscopy can be used for monitoring protein denaturation and associated conformation change processes in a feasible and effective way.

Effects of acid and alkali on the light absorption, energy transfer and protein secondary structures of core antenna subunits CP43 and CP47 of photosystem II

The effects of acid and alkali treatment on the light absorption, energy transfer and protein secondary structure of the photosystem II core antenna CP43 and CP47 of spinach were investigated by the absorption spectra, fluorescence emission spectra and ciruclar dichroism spectra. It has been found that acid treatment caused the appearance of absorption characteristic of pheophytin a (Pheo a), whereas alkali treatment induced a new absorption peak at 642 nm. The energy transfer between β‐carotene and chlorophyll a (Chl a) in CP43 was easily disturbed by alkali, whereas in CP47 was readily affected by acid. As to the effects on the secondary structure of proetins in CP43 and CP47, effects of acid were far less than those of alkali. Both acid and alkali disturbed the microenvironment of Chl a and interfered exciton interaction between Chl a molecules. It was suggested that acid and alkali affect the light absorption, energy transfer and protein secondary structure of CP43 and CP47 in a differenty way. H+ can permeate into the internal space of α‐helix, change Chl a into Pheo a and disturb the microenvironment of pgiments without damaging the secondary structure of protein, whereas OH− can induce the protein unfolding at first, then saponify Chl a to chlorophyllide and disturb the microenvironment of pigments.

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.

Molecular cloning, in vitro expression and enzyme activity analysis of violaxanthin de-epoxidase from Oryza sauva L.

The violaxanthin de-epoxidase gene was cloned from rice (Oryza sauva subsp japonica). The full length of the cDNA is 1887 bp, encoding a 446-amino acids protein with the transit peptide of 98 amino acids. The bacterial expression vector pET-Rvde was constructed and the expression quantity of the exogenous protein increased with the induction time by 0.4 mmol/L IPTG. Its molecular weight was similar with that of the native VDE. Western blotting indicated that the expressed protein has immunological reaction with the VDE polyclonal antibody. The absorbance spectrum together with xanthophyll pigments quantification by HPLC demonstrated that the expressed VDE has its enzyme activity, which can de-epoxidate violaxanthin into antheraxanthin and zeaxanthin in vitro.

Effects of different aggregation forms of LHC II from Bryopsis corticulans on the excited state properties of Chl a

Steady-state and time-resolved fluorescence spectroscopies have been used to study the excited state properties of Chl a in different aggregation forms of light-harvesting complex II (LHC II) from an intertidal green alga, Bryopsis corticulans, i.e. LHC II monomer, trimer and oligomer. When either Chl a or Chl b was selectively excited, the observed decrease in Chl a fluorescence in the oligomer is proved to be caused mainly by the fast fluorescence quenching among Chl a molecules, rather than by the decrease in Chl b-to-Chl a singlet excitation transfer efficiency. Analyses of the picosecond time-resolved fluorescence kinetics identified two exponential decay components in all of the three forms of LHC II: a longer-lived component (4.1–4.7 ns) originating from fluorescence emission of Chl a, and a shorter-lived one (135–540 ps) from the rapid equilibration of singlet excitation among Chl a molecules. The time constant of excitation equilibration is 135 ps in oligomer, 520 ps in trimer and 540 ps in monomer. These results imply that LHC II in oligomer form is inherently able to quench Chl a excitation, a mechanism which may be related to the photoprotection of PS II via changing the degree of LHC II aggregation in Bryopsis corticulans.

The composition and spectral properties of three different forms of light-harvesting complex II

Different aggregates of LHC II play a very important role in regulating the light absorption and excitation energy transfer of plant. Trimeric LHC II was purified from spinach thylakoid membrane. In order to obtain the dimeric and monomeric LHC II, the trimer was treated with the mixture of 2% OGP and 10 μg/mL PLA2, then loaded onto the sucrose density gradient in the presence of 0.06% triton X-100. The LHC II trimer, dimer and monomer isolated by sucrose density gradient all contained three polypeptides with molecular weight of 29, 28 and 26 kd respectively. The pigment composition showed much difference in the content of Chl b and xanthophyll among three forms of LHC II. To study the light capture and excitation energy transfer in different forms of LHC II, the absorption and fluorescence spectra were analyzed. The results clearly showed that the efficiency of energy absorption and transfer was different in the three kinds of LHC II, the highest for trimeric LHC II, intermediate for dimeric LHC II, and the lowest for monomeric LHC II. It was suggested that there might be a physiological homeostasis of different aggregates of LHC II in plants, which is significant for the plant self-regulating upon exposure to variable light environment.

Mg2+-induced LHC II aggregation in thylakoid membrane

Mg2+-induced changes in 77K fluorescence spectra of wheat thylakoid membranes were analyzed by a Gaussian deconvolution program. All spectra were fitted well with 7 Gaussian sub-bands. The sub-band areas for the LHC H macroaggregate (F699) and PS II (F685 and F695) were increased and those for the LHC II trimer (F681) and PS I (F729 and F740) were decreased by Mg2+. Moreover, it was found that the sub-band area ratio for F699 over F681 was dramatically enhanced by Mg2+ by 1.72 times. It was concluded that the LHC II trimer in thylakoid membranes could be aggregated and transformed into its macroaggregate by Mg2+ on a large scale. The possible implication of the Mg2+-induced LHC II aggregation was also discussed.

Relation between artificial electron acceptors and heterogeneity of photosystem II

IN recent years, evidence has been accumulated for the existence of some photosystem Ⅱ(PS Ⅱ) reaction centers which are incapable of the electron transfer from Q A to Q B . These PSⅡ centers are called the inactive PSⅡ centers, compared with the normal ones which are called the active PSⅡ center. Two artificial electron acceptors, 2, 6-dichloro-p-benzoquinone (DCBQ) and 2, 5-dimethyl-p-benzoquinone (DMQ), were often used in studying the heterogeneity of the active and inactive PSⅡ centers. Some believed that DCBQ accepted the electron from both the active and inactive PSⅡ centers and DMQ only from the active PSⅡ cen-...