Gene-specific trans-regulatory functions of magnesium for chloroplast mRNA stability in higher plants

In higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulated via gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions. psbA mRNA (D1 protein of photosystem II), rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNA(His) gain stability at specific magnesium concentrations in an in vitro degradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulated psbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg(2+) rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability.     

Chloroplast mRNA 3'' end processing requires a nuclear-encoded RNA-binding protein.

The protein coding regions of plastid mRNAs in higher plants are generally flanked by 3' inverted repeat sequences. In spinach chloroplast mRNAs, these inverted repeat sequences can fold into stem-loop structures and serve as signals for the correct processing of the mature mRNA 3' ends. The inverted repeat sequences are also required to stabilize 5' upstream mRNA segments, and interact with chloroplast protein in vitro. To dissect the molecular components involved in chloroplast mRNA 3' end processing and stability, a spinach chloroplast protein extract containing mRNA 3' end processing activity was fractionated by FPLC and RNA affinity chromatography. The purified fraction consisted of several proteins and was capable of processing the 3' ends of the psbA, rbcL, petD and rps14 mRNAs. This protein fraction was enriched for a 28 kd RNA-binding protein (28RNP) which interacts with both the precursor and mature 3' ends of the four mRNAs. Using specific antibodies to this protein, the poly(A) RNA-derived cDNA for the 28RNP was cloned and sequenced. The predicted amino acid sequence for the 28RNP reveals two conserved RNA-binding domains, including the consensus sequences RNP-CS1 and CS2, and a novel acidic and glycine-rich N-terminal domain. The accumulation of the nuclear-encoded 28RNP mRNA and protein are developmentally regulated in spinach cotyledons, leaves, root and stem, and are enhanced during light-dependent chloroplast development. The general correlation between accumulation of the 28RNP and plastid mRNA during development, together with the result that depletion of the 28RNP from the chloroplast protein extract interferes with the correct 3' end processing of several chloroplast mRNAs, suggests that the 28RNP is required for plastid mRNA 3' end processing and/or stability.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.     

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.     

Changes in the synthesis of rubisco in rice leaves in relation to senescence and N influx

BACKGROUND AND AIMS: The amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) synthesized in a leaf is closely correlated with N influx into the leaf throughout its lifetime. Rubisco synthesis and N influx are most active in the young leaf during expansion, but are very limited in the senescent leaf. However, it is not established whether Rubisco synthesis can be observed if N influx is increased, even in a very senescent leaf. This study first investigated changes in the relationships between rbcS and rbcL mRNA contents and Rubisco synthesis per unit of leaf mass with leaf senescence. Next, leaves were removed during late senescence, to examine whether Rubisco synthesis is re-stimulated in very senescent leaves by an increase in N influx. METHODS: Different N concentrations (1 and 4 mm) were supplied to Oryza sativa plants at the early (full expansion), middle and late stages (respectively 8 and 16 d after full expansion) of senescence of the eighth leaf. To enhance N influx into the eighth leaf 16 d after full expansion, all leaf blades on the main stem, except for the eighth leaf, and all tillers were removed and plants received 4 mm N (removal treatment). KEY RESULTS: Rubisco synthesis, rbcS and rbcL mRNAs and the translational efficiencies of rbcS and rbcL mRNAs decreased with leaf senescence irrespective of N treatments. However, in the removal treatment at the late stage, they increased more strongly with an increase in N influx than in intact plants. CONCLUSIONS: Although Rubisco synthesis and rbcS and rbcL mRNAs decrease with leaf senescence, leaves at the late stage of senescence have the potential actively to synthesize Rubisco with an increase in N influx.