Barbara van Cleve

Storage, mobilization and interrelations of starch, sugars, protein and fat in the ray storage tissue of poplar trees

The seasonal pattern in starch, various sugars, protein, and fat, and their interrelationship, has been followed in 3-year-old branch wood of poplar trees (Populus x canadensis Moench ‘robusta’) under natural site conditions. The deposition of starch, protein and fat proceeds at different times. Starch accumulates from May until October, fat mainly during the summer months, and protein when the leaves are yellowing in September and October. The maximum concentrations in the branch wood were 15–18 μg starch, 6–9 μg protein, 4–8 μg fat, 10–15 μg sucrose, and up to 30 μg total sugars per milligram dry weight (DW). During starch deposition periods no increased sucrose level is found in the tissue. The maximum daily starch deposition rate was 0.2–0.4 μg starch/day/mg DW of wood. During starch hydrolysis in late autumn and winter, a dramatic increase in sucrose and its galactosides is measured (up to 15–27 μg/mg DW in total). In early spring, before budbreak, the concentrations of these sugars diminishes sharply. In contrast to this clear-cut starch-to-sugar conversion in autumn no significant starch-to-fat conversion is detected. An elevated content of free glycerol, however, is found in winter. In spring, starch and storage protein are mobilized completely, or almost completely, in poplar twig wood. A noteworthy pool of maltose is found transiently during autumn (up to 8 μg/mg DW) and again in spring. The results demonstrate that the individual storage materials, e.g. starch, protein, and fat, are accumulated fairly independently in the wood storage parenchyma. Tissue sugar levels, in contrast, appear to be closely related to the seasonal variations in starch content, on the one hand, and to the acclimation and deacclimation of the cells, on the other. The interrelations of the storage materials and sugars are discussed.

Biochemical, immunochemical, and ultrastructural studies of protein storage in poplar (Populus × canadensis robusta wood

The seasonal changes in protein content have been followed in the wood of Populus × canadensis Moench ‘robusta’, both biochemically and electronmicroscopically at the cellular level. In the storage-parenchyma cells of the twig wood, 4–6 μg · mg−1 DW protein were deposited in the fall, parallel to the yellowing of leaves, and mobilized completely again during the outgrowth of buds in the spring. Environmental impacts on the leaves, e.g. a fungal attack and mechanical injury by a hurricane, were found to affect protein deposition in the wood considerably. Accumulation of protein bodies in the fall and their disappearance from the cells in the spring proceeded parallel to the changes in protein content measured biochemically, proving that these organelles are the main sites of protein storage in the wood parenchyma cells. Using immunogold labelling and an anti-32-kDa poplar storage-protein antibody the protein bodies were shown to be the exclusive sites of storage of a 32-kDa polypeptide. Transient changes in protein content were also observed during fall and winter. Because these changes coincided with changes in protein-body structure and with changes in the population of vesicles and-or tubular membrane cisternae of the cells, an exchange of nitrogen compounds from the storage pool into the structural protein of membranes possibly takes place during these periods. The structural events observed during proteolysis in spring are very similar to those found in seeds. The possible roles of small cytoplasmic vesicles found within protein bodies during proteolysis and of multimembraneous vacuolar compartments during membrane retrieval are discussed.

Evidence for a general light-dependent negative control of NADPH-protochlorophyllide oxidoreductase in angiosperms

The effect of light on NADPH-protochlorophyllide oxidoreductase and its mRNA has been studied in five different species of dicotyledonous plants, bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuns L.) and mustard (Sinapis alba L.), and in two monocotyledonous plant species, maize (Zea mays L.) and barley (Hordeum vulgare L.). In all these species, illumination of etiolated seedlings led to a rapid decline of both the activity and the content of the enzyme protein. These results indicate that there may be a general light-dependent regulation of the enzyme common to higher plants.

Induction by nitrogen and low temperature of storage-protein synthesis in poplar trees exposed to long days

The synthesis of storage proteins in trees of poplar (Populus x canadensis Moench) could not only be induced by a shift from long-day to short-day conditions but also by either a low-temperature treatment or by nitrogen feeding under continuous long-day conditions. The synthesis of the protein did not depend on the cessation of growth and the formation of a terminal bud. The accumulation of the storage protein was in all cases preceded by a drastic increase in the level of the corresponding mRNA.

Biochemical and Ultrastructural Results during Starch-Sugar-Conversion in Ray Parenchyma Cells of Populus during Cold Adaptation

Summary In wood ray parenchyma cells of Populus x canadensis Moench «robusta» the content of starch, individual sugars, protein and fat were followed biochemically during cold adaptation in fall, parallel to changes seen at their ultrastructural level. After leaf abscission, starch is hydrolysed by amylases. Initially, a large maltose pool is formed that is then converted into sucrose and its galactosides. The lower the temperature the more complete is the starch hydrolysis and its conversion into sucrose, raffinose and stachyose. There was no significant starch-fat conversion during this stage. Instead, fat decreased and glycerol increased. The total sugar content in the wood reached 27 and 35 μg mg -1 DW (in hexose units) at 0 °C and -5 °C, respectively. Sucrose, raffinose and stachyose increased to 19 and 27 μg mg -1 at the same time. This increase corresponded fairly exactly to the loss found in starch content. A sugar accumulation in the ray cell protoplast of 350 to 500 μg mg -1 was computed for the winter stage investigated. Parallel to the accumulation of sucrose and its galactosides, the protoplast becomes enriched with a population of tubular and vesicular smooth ER cisternae. This vesicle formation is still insignificant earlier in fall when the large maltose pool is present. Localization of sucrose and its galactosides in these compartments is therefore indicated. Structural changes observed in protein bodies suggest that they are possibly involved in the delivery of the protein moiety for the vast amount of biomembranes formed during this stage. Both the variations in ultrastructure and in individual sugars illustrate the continuing progress in the ray cell protoplast acclimation during fall.

Seasonal variation of amino acids in the xylem sap of “Populus x canadensis” and its relation to protein body mobilization

SummaryThe seasonal changes in the pattern of 21 amino acids occurring in the xylem vessels of Populus twigs have been studied in connection to the mobilization of protein bodies in ray parenchyma cells at the electron microscopic level. Hydrolysis of protein bodies in spring and movement of amino acids into vessels are found to be closely linked. Comprising more than 75% of total amino acid content, glutamine (Gln) is by far the dominant N-constituent of the sap. Gln reaches up to 11 μmol ml-1 in the spring sap while other amino acids only show 1/20 to 1/100 of this amount. From the measured Gln accumulation rates in the vessels in nature and in the vessels of isolated shoots, a minimum flux rate for Gln of 5.6 pmol cm-2 min-1 is calculated for the ray contact cell/vessel interface. Furthermore, because Gln constitutes 75% of the amino acid content of the sap but only 1.3% of the amino acids in the 32 kDa storage protein of the ray cells in the wood (Clausen and Apel 1991), it becomes evident that most amino acids originating from protein body mobilization do not enter the vessels but are used for Gln synthesis preceding Gln release into the vessels.

Protein bodies in ray cells of Populus x canadensis Moench 'robusta'.

Light- and electron-microscopical investigations revealed distinct intravacuolar protein aggregates of 0.3–0.8 μm in diameter in ray cells of poplar during the dormant season. In semi-thin sections, these bodies showed positive protein staining and enzymatic digestibility with pepsin, indicating their proteinaceous nature. Morphometric measurements showed such protein bodies in 7–13% of the area of the ray-cell lumen. This amount corresponded with the protein content of the wood determined biochemically, e.g. 2.0–5.0 μg·mg-1 dry weight. Polyacrylamide gel electrophoresis of the total protein fraction extracted from wood showed prominent polypeptide species with an apparent molecular weight of 30–32 kilodaltons. The results indicate considerable protein storage in ray cells, especially in the form of protein-storage vacuoles.

Immunochemical localization of a storage protein in poplar wood

Summary A protein of about 32 kilodalton was detected in poplar wood during wintertime that vanished during budbreak. After isolation and purification of this protein an antibody was raised against it. Using the immunoblotting technique the antibody could be shown to react specifically with the protein. At the electron-microscopical level, the protein bodies of the ray cells were found to be the particular sites of labelling when the immunogold method was used.

Pith cells of poplar contain photosynthetically active chloroplasts

Pith cells of young poplar (Populus x canadensis Moench) twigs were found to contain chlorophylls a and b. In addition, it was shown that pith cells also have a considerable amount of light-harvesting chlorophyll a/b protein (LHCP), which was identified by Western blotting and localized by immunogold labelling of ultrathin sections. The data strongly indicate that these cells, though they are completely covered by wood and bark and thus are accessible only to very low amounts of light, possess a functionally active photosynthetic apparatus. Evidence for this was found by feeding isolated longitudinal sections of pith with radioactively labelled carbon dioxide and exposing them to light. After incubation, reduced carbohydrates could be detected by thinlayer chromatography, indicating that photosynthesis occurs.

Immunochemical localization of a willow storage protein with a poplar storage protein antibody

SummaryAntibodies raised from a 32 kDa storage protein of poplar wood are found to bind specifically to protein bodies in willow wood ray cells when the immunogold method is used. A polypeptide of ca. 32 kDa is also obtained from the willow wood which reacts with the poplar storage protein antibodies when the immunoblotting technique is used. The results indicate that willow possesses a storage protein which appears closely related to that of poplar.