Studies On Function Of Over-expression Of Plastid-specific Ribosomal Protein 3 In Arabidopsis Thaliana

Ribosome is an organelle that syntheses proteins effectively and precisely according to the instructions of mRNA.Chloroplast ribosome is specific to plant,in which the function of individual proteins is not clear so far.Chloroplast ribosome comprises of 59 proteins,most of which are encoded by nuclear genes with the rest 6 encoded by chloroplast genes.Functional studying of these nuclear encoded chloroplast proteins will expand our knowledge of how ribosome is functioning and regulated at molecular levels. For this purpose,we have constructed the overexpression and suppression vectors of Psrp-3 for the use of sense and antisense study,transformed into Co10 Arabidopsis,and had a detailed study of transgenic plants.The main results are summarized as follows:Psrp-3 is one of specific ribosomal proteins encoded by nuclear gene and then translocated into the chloroplast.Overexpression of Psrp-3 in Arabidopsis significantly enhanced the growth rate.Therefore,we chose four transgenic plants,named L1,L3,L4, L5,which have gradient growth rates in our study,with leaf areas 1.37,1.5,1.46,1.16 timese;fresh weight 1.1,1.27,1.29,1.18 times;dry weight 1.12,1.34,1.32,1.21 times of wild type,respectively.RT-PCR results indicated that the growth rate positively correlated with the expression level of Psrp-3.The same result was obtained at protein level,indicating the increase in biomass is due to the expression of Psrp-3.To further investigate what causes the increase in biomass,first we measured the chlorophyll fluorescence induction kinetics.The result showed that Fv/Fm was unchanged in the transgenic plants,that is to say,photosystem has not been damaged, consistent with the changes of pigment contents.The photochemical quenching in transgenic plants was higher than wild-type,indicating the electron transport activity in transgenic plants was stronger than the wild-type;while the non-photochemical quenching was correlated with PSRP-3 level negatively,i.e.NPQ of L1,L3,L4 and L5, was 34.5,19.1,13.4,18.8%of wild-type.This result indicated that the less the heat dissipation,the more the photosynthesis efficiency,therefore biomass increases.We next examined net photosynthetic rate,transpiration rate and stomatal conductance,and the results showed all of them were correlated with PSRP-3 level positively.Then we wondered if the effect of Psrp-3 is related to the development of the chloroplast.With this in question,we observed the chloroplast ultrastructure.Both transgenic plant and wild-type chloroplasts displayed well-developed membrane systems composed of grana connected by the stroma lamellae,but the number of discs per grana stack in transgenic plant was evidently higher than wild-type,9±2.5 in wild-type, however,17±2.0 in transgenic plant.They are also orderly and compactly arranged in transgenic plant.To gain insight into the steady state levels of the thylakoid proteins in transgenic plants,immunoblot analyses were performed with the antibodies raised against specific subunits of the photosynthetic protein complexes.The results showed that levels of the tested thylakoid proteins per unit of chlorophyll,including CP43,CP47(PSⅡproteins), LHCⅡ,PsbO,PsaA/B of the PSI reaction center proteins,cytochrome f of the cytochrome b6f complex,and CF1βof ATP synthase,were not altered in the transgenic plants.BN-PAGE studies showed that the abundance of PSⅠ,PSⅡ,and LHCⅡprotein complexes were not changed in the transgenic plant.CP43-free PSⅡwas slightly less than wild-type,suggesting the PSⅡcomplex was more stable.We also found that the transgenic plant was sensitive to glucose.In the wild-type, dramatic glucose-responses such as inhibition of chlorophyll synthesis and developmental arrest of leaves were observed when plants were grown on medium containing 7% glucose.However,the transgenic plant showed similar glucose-responses when the glucose concentration in the medium was raised to more than 4%,indicating that the sensitivity to glucose in the transgenic plant may be due to hypersensitivity to ABA.Therefore,we propose that Psrp-3 functions to positively up-regulate photosynthesis at both physiological and developmental levels.