Jörg J. Sauter

Xylem dysfunction during winter and recovery of hydraulic conductivity in diffuse-porous and ring-porous trees

Xylem embolism in winter and spring as well as the occurrence of positive xylem pressure were monitored in several diffuse-porous and one ring-porous tree species (Fraxinus excelsior). In Acer pseudoplatanus and Betula pendula embolism reversal was associated with positive (above-atmospheric) xylem pressures that frequently occurred during a 2-month period prior to leaf expansion. In Acer high stem pressures were occasionally triggered on sunny days after a night frost. The other species investigated showed no positive xylem pressure during the monitoring period in 1995. Populus balsamifera exhibited a complete embolism reversal in 1994, but, like Fagus sylvatica, recovery was slow and incomplete in 1995. Fraxinus did not refill embolized vessels, but relied entirely on the production of new earlywood conduits in May. Populus × canadensis Moench “robusta” did not recover from embolism during the monitoring period. Under a simulated root pressure of 20 kPa however, excised branches of Populus × canadensis restored maximum hydraulic conductance within 2 days, illustrating the great influence of even small positive pressures on cnductivity recovery in spring. In the absence of positive pressure there was no substantial refilling of embolized vessels within a rehydration period of 9 days.

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.

Temperature-induced Changes in Starch and Sugars in the Stem of Populus × canadensis «robusta»

Summary Stem sections of Populus × canadensis «robusta» were taken at the beginning of the dormant season and subjected to temperatures of 10°, 5°, 0°, and −2 °C. Changes in starch content, sugar content, and in the pattern of individual sugars were followed up to four weeks. A strikingly linear relationship between starch breakdown and temperature is observed. The maximal rate of starch hydrolysis was 0.74 μg d -1 mg -1 DWT. A large pool of maltose was maintained at 5° and 10 °C which diminished, however, parallel to the formation of sucrose and its galactosides at lower temperatures. A linear but inverse relationship between starch content and the combined content of sucrose and its galactosides became apparent between −2° and 5 °C. These sugars accumulated from 13 % at 10 °C up to 69 % of the total sugar content at 0° and −2 °C. The results suggest that the temperature determines two separate events, the starch-maltose conversion and the synthesis of sucrose and its galactosides from the maltose moiety.

Interrelationships between ultrastructure, sugar levels, and frost hardiness of ray parenchyma cells during frost acclimation and deacclimation in poplar (Populus × canadensis Moench ‹robusta›) Wood

Summary Ray parenchyma cells of poplar wood ( Populus × canadensis Moench ‹ robusta ›) were studied for changes in ultrastructure and in the level of various sugars in fall, winter and spring in relation to freezing resistance as measured by LT 50 (temperature at which 50% of the cells are injured). Before acclimation (mid-October), frost hardiness is low (LT 50 −3 to −5 °C), sugar levels have not yet increased while the ray cell protoplast has already become dense in structure. This is due mainly to the elimination of large vacuoles, their replacement by small storage protein vacuoles, and the vast accumulation of storage material in general, i.e. in amyloplasts, protein bodies, and oleosomes. Frost acclimation started during leaf fall (end of October) concomitant with a sudden increase of total sugar content, consisting mainly of sucrose and its galactosides. An increase in these sugars is paralleled at the ultrastructural level by a prominent vesicular and cisternal ER-system originating in the cytoplasm. Full acclimation (LT 50 lower than −50 °C) is obtained within 3 weeks (mid-November) and coincides with maximum content of sucrose and its galactosides (reaching 300 µg mg −1 DW in ray cells) and with a prominent development of vesicular and cisternal ER. The evidence for the accumulation in this elaborated ER-system of these sugars is discussed and a proposal for its functional significance both as an intrinsic device for dehydrating the protoplast during frost hardening and for delivery of membrane material to the plasma membrane is made. Transient rises of winter temperature above freezing were paralleled by a noticeable decrease in frost resistance (LT 50 −40 to −30 °C), declining sugar levels, and by gradual diminution of the particular ER-vesicles in cytoplasm. In March, at temperatures of 5 to 10 °C, sugar content rapidly lowers concomitandy with complete vanishment of vesicles from the cells. A decrease in frost resistance, in contrast, begins later, e.g. in mid-April, suggesting the involvement in frost hardiness of non-sugar compounds at this stage.

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.

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.

Seasonal changes of sucrose-phosphate synthase and sucrose synthase activities in poplar wood (Populus × canadensis Moench ‘ robusta ’) and their possible role in carbohydrate metabolism

Summary Two important enzymes of sucrose metabolism, sucrose-phosphate synthase (SPS, EC 2.4.1.14) and sucrose synthase (SuSy, EC 2.4.1.13), were investigated in the ray parenchyma cells of the trunk wood of Populus × canadensis Moench ‘robusta’ throughout the year. The activity of SPS increases dramatically in autumn in parallel with leaf fall, reaches a maximum level in winter at the time of the starch-to-sugar conversion and declines in spring during starch resynthesis and mobilisation. In summer, the activity of SPS remains at a very low level. These seasonal changes in SPS activity were identical both under Vmax- and under Vlim-assay conditions. In temperature-controlled storage experiments with twig sections, the activation state of SPS, termed as Vlim/Vmax × 100, was substantially higher after storage at -5 °C in contrast to storage at +10 °C. A Western blot analysis, using a polyclonal antibody, revealed a molecular weight of about 130 kD for the SPS-polypeptide in poplar wood with highest levels of SPS enzyme protein in winter and lowest levels in summer. SPS of other tree species (Acer, Fagus, Salix) exhibited a molecular weight in a similar range. The activity of SuSy started to increase in late autumn, was high in winter and declined in spring. In contrast to SPS, SuSy shows a remarkably high activity in the outer wood area in summer while it remained low in the middle and inner area of the trunk wood. This high SuSy activity correlates with the differentiation of the xylem cells rather than with starch deposition. The significance of the SPS in autumn and winter for the starch-to-sugar conversion during cold adaptation of xylem parenchyma cells and of the SuSy for wood formation processes is discussed.

Changes in the Partitioning of Carbohydrates in the Wood during Bud Break in Betula pendula Roth

Summary Changes in the carbohydrates of the wood have been investigated during the growth and blossoming of catkins in branches of Betula pendula Roth collected in late winter and subjected to various temperature treatments (0°, 10°, and 21 °C). These changes were followed separately for the apoplast and for the symplast. The growth and blossoming of catkins was paralleled by a prominent resynthesis of starch (e.g. from 3.0 to 7.7 µmg−1 dwt) and a great decrease of sugars in the symplast (e.g. from 20.2 to 6.4 µgmg−1 dwt), a remarkable release of sugars into the apoplast (maximum content 8.98 gl−1), a steep rise in transpiration, and a sharp drop in the pH of the xylem sap (e.g. from 7.5 to 5.7). In the symplast, the ratio of starch to sugars changed from ca. 1:6 to 1:1 within 10 days while its sugar content decreased from 0.65 M to 0.2 M. During catkin growth and blossoming, the carbohydrate content of the branch wood decreased by about 50%. Because the amount of transpired water and its sugar content were found to account almost fully for the observed loss of carbohydrates from the symplast, the xylem pathway is attributed an essential role in translocating organic material in the early stage of spring. In the symplast, fructose, glucose, maltose, sucrose, and its galactosides raffinose and stachyose are abundantly found at 0°C with the contents lying between 2.3 and 7.2 µg mg−1 dwt. In the apoplast, in contrast, 97 to 98 % of the sugars are made up by fructose, glucose, and sucrose at the same time. Considerable changes in the absolute and the relative content of individual sugars are observed in the symplast during the storage periods. The physiological significance of the observed changes in carbohydrates, and of the xylem pathway are discussed.