Metabolism of proteins in developing leaves of oats (Avena sativa L.)

English. The development of the first leaf of oats ( Avena sativa L.) comprises four phases. About 4 day after sowing the leaf emerges and starts to expand. Expansion is complete and maturity is reached at 7 to 9 days. Thereafter, senescence ensues, as expressed by a gradual loss of chlorophyll over the period from 12 to 37 days. Finally the leaf withers, and dies by about 37 days.RNA and protein contents start to decrease before the onset of chlorophyll loss, and during the phase of senescence all three parameters decline in a coordinated manner. Since proteins are essential to the functioning of the leaf, their loss is considered to be a determining factor in the rate at which senescence proceeds. However, inhibitors of protein synthesis have been shown to block loss of protein and chlorophyll, indicating that also synthesis of proteins is required for senescence to progress. These proteins are not major proteases, nor are they likely to be membrane-associated, because membrane systems remain intact until a late stage of senescence.The present study was undertaken to search for proteins that are specific to each of the developmental stages in the life of the leaf, and to determine specific properties of proteins that function particularly during the stage of senescence. Changes in the pattern of soluble proteins during leaf development were followed by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (Chapter 2). Phenol-soluble protein samples were separated in up to 500 spots by isoelectric focusing between pH 5 and 8 in the first dimension and 12.6% SDS slab gel electrophoresis in the second dimension. The gels were stained with silver. A number of 122 protein spots were further characterized by their presence in isolated chloroplasts. The large and the small subunits of ribulosebisphosphate carboxylase/oxigenase (Rubisco) were identified by immunoblotting.Major changes occurred during the period of leaf expansion: 39 mostly low-molecular-weight polypeptides disappeared and 34 mostly high-molecular-weight spots became apparent. Of these, 16 and 13, respectively, were associated with chloroplasts. During the subsequent loss of protein and chlorophyll, indicative of senescence, the number of spots decreased gradually without new polypeptides becoming apparent, 75% of the total number being still detectable in yellow, 37 days-old leaves. About half of the disappearing spots were lost before the leaf had expanded to its final length. No polypeptides were detected that were present exclusively in the later stages of leaf development: maturity, senescence, and final collapse.It was considered that such proteins might be present in amounts below the limit of detection by general protein staining, either because they are synthesized in very small amounts, or because they are subject to rapid turnover. To be able to follow the synthesis and degradation of individual proteins, while avoiding effects of leaf detachment or wounding, a method was developed to label the proteins at different stages of development of intact leaves attached to the plants (Chapter 3). The following methods were tested: growth of seedlings on 35S-sulfate-containing Knop medium, labelling with 35S-methionine by vacuum infiltration of the leaf, injection into the leaf base or into the seed near the embryo, or wiping the surface of the leaf with ethanol and subsequent incubation in the labelled solution. The first four methods were unsuitable because of insufficient uptake, preferential transport to other plant parts, too much dilution by endogenous aminoacids or radiation damage. A specific activity of 10 5dpm / 20 μg of leaf protein, minimally necessary to obtain a distinct fluorogram of the gels, was reached only upon the treatment with ethanol. Under these conditions no more than 170 kBq of 35S-methionine was required in the uptake solution.When the 4.5 cm distal parts of the first leaf were thus labelled for 12 h at different stages of development, similar amounts of label were retained in the segments, but incorporation into protein decreased from 28% in 7-days- to 3% in 27-days-old plants. Even at a late stage of senescence a great many proteins were still being synthesized. Synthesis of Rubisco and the other chloroplastassociated proteins declined more rapidly than general protein synthesis. Although Rubisco ceased to be synthesized by 13 days, it was still clearly present in 27-days-old leaves indicating that, once synthesized, the protein is very stable. During senescence two sets of relatively high -molecularweight proteins became more pronounced. In addition, three proteins with a molecular weight around 67 kDa and isoelectric points between 6.5 and 6.8 became the most prominently synthesized proteins in older leaves. Since these senescence-associated proteins were hardly visible on silver-stained patterns, they seem to be subject to rapid turnover. A functioning of these proteins in senescence could explain the requirement of protein synthesis for senescence to proceed (Chapter 4).By labelling distal leaf parts of 7- and 15-days-old plants for 24 h and following the loss of label from individual protein spots, the rates of degradation of the synthesized proteins were determined over a 14-day period. Of the about 300 spots that could be distinguished, a large number turned over quickly (disappearing spots), while a smaller number was degraded very slowly (persisting spots). Thus, protein half-lifes varied from a few days to over at least a week. This pattern of in vivo breakdown was compared to that in vitro in soluble-protein extracts at pH 5.5 and 7.6, the optimal pH values of the acidic and neutral proteases, respectively. Although 75% of the proteins were degraded similarly in vivo and in vitro , substantial changes in the relative rates of degradation of the other proteins occurred under the different conditions. Notably, Rubisco was degraded very slowly in vivo , but rather quickly and in a different way in vitro at pH 5.5. As it has been found that there is no relationship between the level of protease activity and the rate of protein degradation in vivo, these results support our previous conclusion (Van der Valk and Van Loon 1988) that the proteases and their protein substrates are spatially separated in vivo . Whereas rapidly turning-over cytoplasmic proteins might be imported into the vacuole for degradation, long-lived chloroplastic proteins, such as Rubisco, are likely to be degraded by proteases within the organelle.To investigate in how far the changes observed in attached leaves also occur upon detachment, distal leaf segments were incubated vertically with their bases in water. Senescence of detached leaves in light did not differ significantly from senescence in attached leaves. In darkness, protein was lost at a higher rate, but several proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight and senescence-associated proteins increased in amount. Additional changes associated with incubation in light or darkness were not related to senescence. The cytokinin benzyladenine delayed and abscisic acid accelerated the changes compared to water. Besides changes previously identified in leaves senescing on the plant, detached leaves showed alterations that reflect their condition of incubation rather than their developmental stage. For this reason, detached leaves are less suitable for the study of senescence than leaves attached to the plant (Chapter 6).The comparison of many protein patterns was facilitated by the development of a computer program, GELSCAN, for the qualitative matching of any pair of gels. The program is written in PASCAL and makes use of a 640 kB personal computer. This program is a valuable tool for an objective analysis of 2D-PAGE patterns (Chapter 7).Finally, the regulation of protein synthesis and degradation during development of the first leaf of oats is discussed (Chapter 8). No specific properties of the proteins themselves, as identified by 2D-PAGE, act as determinants of their rate of degradation. The high-molecular-weight proteins, and particularly the senescence-associated proteins, are synthesized in increasing amounts during senescence and may play a role In mechanisms governing the senescence progress.

Dutch; Flemish. De ontwikkeling van het eerste blad van haver is te verdelen in 4 fasen. 4 dagen na het zaaien komt het blad te voorschijn en begint te strekken, totdat het tussen 7 en 9 dagen volgroeid is. Daarna begint het blad te verouderen, hetgeen zich uit in een geleidelijk verlies van chlorofyl over een periode van 12 tot 37 dagen. Tenslotte verwelkt het blad, droogt uit en sterft af rond dag 37. In het onderzoek is gezocht naar eiwitten die specifiek zijn voor elk van de ontwikkelingsstadia tijdens het bladleven. Daarnaast is getracht specifieke eigenschappen te definieren van eiwitten, in het bijzonder voor het functioneren tijdens het verouderingsstadium. Veranderingen in het patroon van de oplosbare eiwitten gedurende de bladontwikkeling werden gevolgd m.b.v. 2-dimensionele polyacrylamide-gel-elektroforese. Om de synthese en afbraak van individuele eiwitten te volgen, maar tegelijk de effecten van afsnijding of verwonding te vermijden, werd een methode ontwikkeld om de eiwitten in intacte bladeren aan gehele bladeren in verschillende ontwikkelingsfasen te merken met een radioactieve precursor