Caution in the interpretation of plant ribosome studies (<i>Short Communication</i>)

Leaver, Christopher J.; Dyer, John A.

doi:10.1042/bj1440165

Cited by 30 publications

(16 citation statements)

References 8 publications

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“…A likely explanation for this discrepancy is the manner of preparation of the crude polysome fraction. A common method of isolating polysomes from plant tissue involves centrifugation of the initial crude extract through a dense sucrose cushion (20,28,29). This concentrates the polysomes, monosomes, and free messenger-ribonucleoproteins and separates them from debris that can interfere with their subsequent analysis.…”

Section: Resultsmentioning

confidence: 99%

“…This concentrates the polysomes, monosomes, and free messenger-ribonucleoproteins and separates them from debris that can interfere with their subsequent analysis. A major problem with this sedimentation procedure, especially when using relatively short centrifugation times, is that it tends to enrich for polysomes while leaving monosomes and free messenger-ribonucleoproteins underrepresented in the pellet (28,29). In the experiment described above ( Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

mRNAs encoding ribulose-1,5-bisphosphate carboxylase remain bound to polysomes but are not translated in amaranth seedlings transferred to darkness

Berry

Carr

Klessig

1988

Proc. Natl. Acad. Sci. U.S.A.

118

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Communicated by C. S. Levings III, February 19, 1988 ABSTRACT When light-grown seedlings of amaranth are transferred to total darkness, synthesis of the large subunit (LS) and small subunit (SS) of ribulose-1,5-bisphosphate carboxylase [RbuP2Case; 3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] is rapidly depressed. This reduction in RbuP2Case synthesis occurs in the absence of any corresponding changes in levels of functional mRNA for either subunit. Four hours after light-to-dark transition little, if any, changes in the distribution of LS and SS mRNAs on polysomes could be detected. The association of these mRNAs with polysomes was authenticated by treatment with RNase A or puromycin. Furthermore, polysomes were able to synthesize LS and SS precursor in cell-free translation systems supplemented with inhibitors of 'initiation. Therefore, during a light-to-dark transition LS and SS mRNAs remained bound to polysomes but were not translated in vivo, suggesting that control is exercised, in part, at the translational elongation step.Ribulose-1,5-bisphosphate carboxylase [RbuP2Case; 3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] is found in the chloroplasts of all higher plants and is a principal enzyme in photosynthetic carbon fixation. This enzyme has a molecular mass of -555,000 and consists of eight large (51-58 kDa) and eight small (12-18 kDa) subunits (LS and SS, respectively) (1), with the active site located on the LS (2, 3). The LS is encoded on the chloroplast genome and translated on 70S chloroplast ribosomes (4, 5). The SS is encoded in the nucleus and translated on free, cytoplasmic ribosomes as a 20-kDa precursor (6-8). The precursor is processed to its final size during transport into the chloroplast, where it assembles with LS polypeptides to form the active holoenzyme.The control of RbuP2Case production and activity in a number of plant species is very complex, with regulation occurring at many levels. Control at the level of LS or SS mRNA accumulation has been well documented (9). In several cases alterations in transcriptional activity have been shown to be responsible for these changes in mRNA levels (9). At the other end of the spectrum, regulation at the posttranslational level via turnover of the protein (10) as well as by activation (1, 11) or inhibition (12) of the enzyme's activity has also been reported.We have previously described the effects of environmental (light) (13,14) or developmental (13, 15) signals on the expression of LS and SS genes in the C4 dicotyledonous plant Amaranthus hypochondriacus. The expression of these genes is regulated not only at the level of mRNA accumulation but also posttranscriptionally. In amaranth cotyledons rapid and dramatic alterations in the synthesis of the LS and SS polypeptides occur in response to changes in illumination without corresponding changes in mRNA levels. Because the stability of these polypeptides and the functionality in vitro of their respective mRNAs were not affected by alterations in illumination, th...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mRNAs encoding ribulose-1,5-bisphosphate carboxylase remain bound to polysomes but are not translated in amaranth seedlings transferred to darkness

Berry

Carr

Klessig

1988

Proc. Natl. Acad. Sci. U.S.A.

118

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“…When material was sedimented through a cushion at 100,000g, the trends obtained following stress were the same, but the polyribosome percentages were exceptionally high. This is now known to result from partial sedimentation of the monoribosomes (11).…”

Section: Methodsmentioning

confidence: 99%

Water Stress, Rapid Polyribosome Reductions and Growth

Rhodes

Matsuda²

1976

Plant Physiol.

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Measurements of the water status of various plat tissues exposed to differing levels of salts for 1 hour were made using the recently developed Campbell J-14 press (Logan, Utah). Values obtaied with the press were found to correlate well with estimates of relative water content, and experiments with 3-day-old pumpkin seedlings showed that detectable changes in press values of cotyledon tissues could be obtained within 5 minutes folowing salt-or desiccation-induced stress.Pobyiosome levels were measured in tissues from various plant species following short duration water stress. A small reduction in

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“…Furthermore, the usually employed, subsequent ultracentrifugation of 2-6 h at 100000 x g recovers selectively most of the polyribosomes, while a significant amount of the monoribosomes and the subunits remain in the supernatant. Longer periods of centrifugation, on the other hand, increase the rate of degradation of the polyribosomes [37-401. The loss of polyribosomes can be substantially reduced to about 10-20';/,, if a suitable detergent, such as deoxycholate or Triton X-100, is included in the homogenization buffer [37,39]. Since deoxycholate has been shown to remove elongation factors, among other proteins, from the polyribosomes and to inhibit the cell-free system [41-431, we chose Triton X-I00 for our studies of the effects of a detergent on the isolation procedure for the polyribosomes.…”

Section: The Cell-free Systemmentioning

confidence: 99%

Light‐Induced Enzyme Synthesis in Cell Suspension Cultures of Petroselinum hortense

Schröder

Betz

Hahlbrock

1976

European Journal of Biochemistry

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The conditions for protein synthesis in vitro with polyribosomes from cell suspension cultures of parsley (Petroselinum hortense) and a wheat-germ extract were investigated. Two different criteria were used as estimates of the translational activity: (a) the total rate of incorporation of [35S]methionine into acid-insoluble material; (b) the ratio of large (molecular weight > 25 000) to small (molecular weight <25000) peptide products. Depending on which of the two criteria was employed, the pH optimum and the optimal concentrations for Tris-acetate, magnesium acetate, KCl, methionine and the wheat-germ extract differed considerably. The translational activity of the polyribosomes (both criteria) was efficiently protected by 0.1 M MgZf against degradation during the isolation procedure.The rate of synthesis of phenylalanine ammonia-lyase in vitro with the polyribosomes was determined by measuring the incorporation rate of ~-[~~S]methionine into protein which was precipitable by a rabbit antiserum preparaed for the purified enzyme. The immunoprecipitate was analyzed by disc gel electrophoresis in the presence of dodecylsulfate and was shown to contain small amounts of the complete enzyme subunits and relatively large amounts of shorter peptides which were also characteristic for the enzyme.The time courses of light-induced changes in the rate of phenylalanine ammonia-lyase synthesis in vitro were investigated during a period of 15 h under two different conditions of induction: the cell cultures were irradiated with ultraviolet light either (A) continuously or (B) for 2.5 h and then returned to darkness. Although the highest rate of enzyme synthesis was observed somewhat later in experiment A than in experiment B, the periods of time during which the rate of synthesis increased rapidly were limited in both cases to only a few hours.The results obtained in vitro were identical within the limits of the experimental error with theoretical calculations of the changes in the rate constant of phenylalanine ammonia-lyase synthesis in vivo. These changes were calculated from the corresponding curves for the changes in the enzyme activity under the two conditions of induction. The results are in agreement with previous observations suggesting that the induction of phenylalanine ammonia-lyase by light in the parsley cells was a shortterm effect whose efficiency was greatly reduced within the 15 h of experimentation, even under continuous irradiation.Phenylalanine ammonia-lyase catalyzes the conversion of phenylalanine to cinnamic acid and is the first of a sequence of more than 10 enzymes which are involved in the light-induced formation of flavone

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Caution in the interpretation of plant ribosome studies (Short Communication)

Abstract: Published methods ofribosome isolation from plant tissues result in a partial and selective recovery of the total ribosome population. The enrichment of polyribosomes obtained may give a false impression of the change in translational capacity of the tissue under study.

Cited by 30 publications

References 8 publications

mRNAs encoding ribulose-1,5-bisphosphate carboxylase remain bound to polysomes but are not translated in amaranth seedlings transferred to darkness

mRNAs encoding ribulose-1,5-bisphosphate carboxylase remain bound to polysomes but are not translated in amaranth seedlings transferred to darkness

Water Stress, Rapid Polyribosome Reductions and Growth

Light‐Induced Enzyme Synthesis in Cell Suspension Cultures of Petroselinum hortense

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