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.

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 3∶1 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, δ-aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The.precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translo- cation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with S-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seed- lings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurna1 fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a nove1 regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.

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.

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.

The 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.

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.

During the dormant period and especially during budbreak a poplar storage protein could be detected in xylem sap on SDS-PAGE and immunoblot; after budbreak it was absent. The permeability of poplar vessels and pit membranes for proteins larger than the poplar storage protein was confirmed, giving evidence for a long distance transport of entire storage proteins via the transpiration stream.

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.

The seasonal changes in protein content have been followed in the wood of Moench ‘’, both biochemically and electronmicroscopically at the cellular level. In the storage-parenchyma cells of the twig wood, 4–6 μg · mg 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.

Antibodies 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.

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.