Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate Carboxylase

Abstract

Ribulose bisphosphate carboxylase from Lemna minor resembles the structure reported for the enzyme from other plants. When grown in the light, the enzyme appears to undergo little or no degradation, as measured by a double-isotope method. This situation is similar to that reported for wheat and barley, but is unlike that reported for maize, where the enzyme degrades at the same rate as total protein. Prolonged periods of darkness usually induce leaf senescence, characterized by the rapid degradation of chlorophyll and protein, with ribulose bisphosphate carboxylase undergoing preferential degradation. In L. minor there is selective protein degradation in the dark, but chlorophyll and ribulose bisphosphate carboxylase are stable when fronds are kept in the darkness for up to 8 days. It appears that Lemna is not programmed to senesce, or at least that darkness does not induce senescence in Lemna. Although there is no evidence for in vivo degradation or modifilcation of ribulose bisphosphate carboxylase during prolonged periods of darkness, extracts from fronds which have been kept in the dark for periods in excess of 24 hours convert ribulose bisphosphate carboxylase to a more acidic form. The properties of the dark-induced system which acts on ribulose bisphosphate carboxylase, suggest that it may be a mixed function oxidase. The proposition that the selectivity of protein degradation is genetically determined, so that the rate at which a protein is degraded is determined by its charge or size, was tested for fronds grown in the light or maintained in the dark. There was no significant correlation between protein degradation and either charge or size, in light or dark.A great deal of evidence has been marshaled to support the view that the key enzyme of photosynthesis, RuBPCase' (EC 4.1.1.39), undergoes little or no degradation prior to leaf senescence. The strongest evidence that RuBPCase does not degrade while it is being synthesized has been presented by Huffaker and his colleagues (19,29). The same group has shown that, during the dark-induced senescence of detached leaves of barley, the initial loss of protein is almost entirely due to the degradation of RuBPCase (28). This behavior has also been observed in the dark-induced senescence of attached wheat leaves (37, 38). The stability of RuBPCase prior to senescence, and its rapid degradation during senescence, has led to its classification as a leaf storage protein ( 19,21 is the radioactivity present in protein immediately after labeling with a radioactive amino acid and P(,) is the radioactivity remaining after time t. This ratio can readily be transformed to give the rate constant of degradation (KD) by means of the equation for first order decay KDt = ln(P(o)/P(,)).Alternatively, degradation can be expressed as a half-life t(o.5) t(O.5) = ln 2/KD. For reasons presented in the "Discussion" we prefer to use the direct isotope ratio, rather than the logarithmic transform.The double-label method (2) has been widely used to test the proposition that t...

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

“…However, RbuP2Case in other systems has been shown to be very stable, with half-lives on the order of days (30,31). In addition, our previous results (14) and those of Ferreira and Davies (32) failed to detect any increased turnover of these polypeptides in the dark. However, we cannot rule out the possibility that two subpopulations of one or both subunits exist-one population that is stable and was detected in our previous pulse-chase experiments and a second population that is rapidly turned over If, as we suspect, translational elongation is blocked in dark-shifted cotyledons, then this block must be readily reversible.…”

Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate Carboxylase

“…Chl a and b were also determined separately, but since the ratio was constant the data are not reported here. L-[ I -'4C]leucine, as described in the previous paper (15 Figure 5. The data confirm the conclusion from Figure 4, with '4C-labeled fronds as previously described (15 Figure 6 should be compared with Figure 4, which depicts the effect of nutrient starvation in light on protein degradation.…”

“…The model can be extended to the degradation of proteins in organelles, provided that the organelles are degraded within the vacuole. Indirect evidence supporting this is that the two major endoproteinases which constitute over 99% of the total endoproteolytic activity of barley leaves (24) (15) by mixing the suitable pair of '4C-and 3H-labeled fronds, except that for the homogenization of fronds stressed in H20 in the dark, the extraction medium used was 100 mM-Tris-HCI buffer (pH 7.5), containing 1 mm phenylmethylsulfonyl fluoride, 2 mM KCN, and 2 mm EDTA, to protect against the action of unspecific oxidases (15).…”

“…Total soluble proteins from Lemna were fractionated in the FPLC Mono Q ion exchange column, in the FPLC Superose 12 gel filtration column or by SDS-PAGE, as previously reported (15). The peaks corresponding to RuBPCase and nucleic acids separated by FPLC and also the subunits of RuBPCase separated by SDS-PAGE were identified as in the previous paper (15). It should be noted that there is some variability in the point at which the RuBPCase is eluted from the Mono Q column.…”

“…It should be noted that there is some variability in the point at which the RuBPCase is eluted from the Mono Q column. Polyacrylamide gel electrophoresis and the measurement of radioactivity in samples were performed as described by Ferreira and Davies (15).…”