Results and Discussion

Results and Discussion

Effects of light and O2 on the mRNA levels of genes for LH，RC proteins，Bchl and Crt biosynthesis.Light intensity and O2concentration significantly effect the levels of mRNA for LH，RC and pigment biosynthesis（see Table 1）.It has been shown that：① A shift to dark causes the decreased expression of photosynthetic genes；②An increase in light intensity results in decreased expression of the genes for LH，RC and Bchl biosynthesis，but it results in increased expression of crt genes；③ O2 greatly reduced the expression of genes for LH，and RC；however，the relative amounts of mRNAs from crt genes rise；④ The mRNA for LH-Ⅱ is more sensitive to O2 than that of the mRNA for LH-Ⅰ，indicating that the genes for LH-Ⅱ and LH-Ⅰ/RC are independently regulated.These findings are supported by Northern hybridization（see Figure 1）.The gene for LH-Ⅱ has one transcript（0.5 kb），whereas the gene for LH-Ⅰ has two transcripts：0.5 kb and 2.6 kb.The 0.5 kb transcript is more abundant and is only complementary to the genes for LH-Ⅰ（β，α），while the 2.6 kb transcript hybridizes to five genes coding for LH-Ⅰ（β，α），RC-L（see Figure 1），RC-M and ORF 02397（data not shown）.The 2.6 kb transcript is more sensitive to O2 than the 0.5 kb transcript（see Figure 1）.The gene for RC-H has two transcripts，1.2 and 1.4 kb.They initiated within ORF F1696，and they are regulated differently by light intensity.

Decay rate of mRNA from photosynthetic genes and O2 regulation.In order to study the effects of O2 on the expression of photosynthetic genes in more detail，we have conducted an O2 shift experiment.R.capsulatus strain SB1003 was grown photosynthetically（see Figure 2，PS），then shifted to LO2（medium/flask=8/10，v/v）（see Figure 2，PS→LO2），or to high 02（medium/flask=0.8/10，v/v）（see Figure 2，PS-O2）.The absorption spectra of pigments shows that the Bchl content（peaks at 360，600 and 770 nm）decreases during the shift.A more detailed time course reveals that the total Crt content（absorption between 400 and 500 nm）increases slightly in the first 3 hours，then decreases（data not shown）.The change in relative ratio of A453 and A484 is due to the oxidation of spheroidene，a major Crt in PS cell，to spheroidenone，a major Crt in aerobic cells，as shown by TLC analysis（data not shown）.This oxidation is catalyzed by a metal-containing oxygenase［14］，encoded by crtA located in BamH fragment［11］.Figure 3 shows the decay rate of mRNAs and the effects of O2.When an inhibitor of transcription，proflavin，is added to cell cultures，the levels of mRNA from all photosynthetic genes decrease.The half-lives of mRNAs for LH-Ⅱ，LH-Ⅰand RC（L，M，H）are 24，19 and 10 min respectively.O2 probably turns off the transcription of genes for LH（Ⅰ，Ⅱ），RC（L，M，H）and Bchl biosynthesis，resulting in a decrease in their mRNAs.O2 appears also to stimulate the degradation of mRNA for LH-Ⅱ.It is interesting to note that the level of mRNA in BamH fragment containing crt A，I，B and bchl increases two fold after O2 shift Such an opposite response to O2 is mainly due to crtA，because a Tn5.7 insertion in crtA（in KZR9A12）abolishes this response（data not shown）.Crt has been long known to have two functions：harvesting light and protecting cell from photooxidative killing［15］.Plants and bacteria have a variety of mechanisms for such protection［16］.Carotenoids，as important constituents of chloroplast membranes，react with singlet O2 and therefore protect chlorophyll and membranes against damage［16］.This is the first report that such protection can be regulated at the level of gene expression under high light and O2.

Cooperation and interdependence of photosynthetic genes.The photosynthetic genes for LH，RC and pigment biosynthesis are interdependent.Mutation in each of the genes for RC-H（UAR87）［17］，Crt（9F4）or Bchl（MB1007）biosynthesis results in significantly reduced amounts of mRNAs from the rest of the genes（see Figure 4）.A longer exposure of the autoradiograph（data not shown）shows that although the mRNA levels in these mutants are very low，they are detectable.A close link exists between genes for LH-Ⅱ and Crt biosynthesis.A comparison of the decay rate of mRNAs for LH-Ⅰ，LH-Ⅱ，RC-L，RC-M and RC-H after treatment with proflavin as transcription inhibitor reveals that these mRNAs in the mutants are at least stable as in wild-type.Therefore，the reduction in mRNA levels in these mutants is due to transcriptionsl control.We suggest that proper and accurate assembly of the photosynthetic compontents in ICM is necessary for transcription.Absence of one of these components causes failure of assembly of the photosynthetic apparatus.Accumulation of these components（either proteins or pigments）may tum off transcription.Futhermore，unassembled proteins may become more susceptible to endogenous proteinase.