Polarity of Xylem Formation in Isolated Stem Segments of Pinus silvestris

The culture of isolated stem sogments was used as a technique to study polarity of xylem formation in Pinus silvestris L. Cambial activity was greatest at the apical or the basal end, whichever received the complete fresh medium, but decreased more abruptly with increasing distance when the basal end was so supplied. Whether gradients of activity increased basipetally or acropetally also depended upon the end to which the auxin and the sugar components were supplied. Ring-barking of segments, then cultur-ing them with apical supply of medium to the xylem, resulted in inhibition of xylem production below, and above, the ring. Cambial activity at the apical ends of such segments was expressible as a logarithmic function of the uninterrupted length of contiguous extraxylary tissues. The involvement of polarity phenomena in control of cambial activity in isolated stem segments is confirmed.     

Auxin Stimulation of Cambial Activity in Pinus silvestris II. Dependence upon Basipetal Transport

Natural auxin content has been determined in the cambial region of large Pinus silvestris L. trees at various dates during the year. The tissue was collected from the stem of intact or ring-barked trees and from stumps remaining after the trees were cut down at breast height in early summer or late autumn. No seasonal decrease of concentration of the extractable auxin in the cambial region could be detected. Decapitation or ring-barking produced severe reduction in auxin content and arrested cambial division. In the next season the auxin level and the cambial activity remained completely depressed. It is concluded that without tissue continuity in the region external to xylem and without basipetal supply of substances, no mechanism operated by roots or remaining stem tissue near the tree base can ensure a high level of auxin in the cambial region or activate and maintain the cambial division. The activity of extracted pine auxin was found not to be identical with the stimulatory potential of authentic IAA determined by standard bioassays. The possibility of interaction with other extracted substances is discussed.     

Auxin Stimulation of Cambial Activity in Pinus silvestris I. The Differential Cambial Response

Isolated stem segments of Pinus silvestris L. produce new xylem in sterile culture for 5 weeks if sucrose and IAA are present in the medium. The response of cambium varies in the course of the season and along the tree stem. The cambium is more sensitive in spring and in the stem portion closer to tree apex than later in the season and closer to the stem base. Spring initiation of cambial activity in adult pine trees under natural conditions could not be correlated with any consistent concentration gradient of natural auxin extracted from the cambial region. Thus, the relation between concentration of auxin and the activity of cambium is complex and involves changes of cambial responsivity. Interaction with gibberellic acid or kinetin and changing concentration of sucrose were studied during the season, but none of these substances alone appeared to be responsible for the observed variation in cambial response to auxin.     

Inhibition and Requirement of Natural Stimulator for Cambial Xylem Production in Isolated Stem Segments of Pinus silvestris

Formation of xylem in sterile culture of isolated pine stem segments supplied with synthetic media continues only for 4–6 weeks. The stem segments originally do not contain an eluable inhibitor affecting elongation in the test of pine hypocotyl sections. After 4 weeks inhibition is detected. Supplying the medium with a methanolic extract from the cambial region of pine stem prolongs xylem production up to 15 weeks. IAA interacts synergistically with a natural stimulator of cambial activity.     

Development of photosynthetic electron transport in Pinus silvestris

Photosynthetic electron transport activity has been measured in chloroplasts isolated from dark-grown seedlings of Pinus silvestris L. and in chloroplasts isolated from seedlings subjected to illumination for periods of up to 48 h. Activities of photosystem 2, photosystem 1 and photosystem 2 plus 1 have been measured. Chloroplasts isolated from dark-grown seedlings showed significant electron transport activity through both photosystems and through the entire electron transport chain from water to NADP. Illumination of the seedlings for only 5 min markedly promoted photosystem 2 activity. The artificial electron donor, diphenylcarbazide. promoted activity in chloroplasts from dark-grown seedlings and in chloroplasts from seedlings illuminated for up to 30 min. In comparison to photosystem 2 and overall electron transport from water to NADP, photosystem 1 activity increased only slightly during illumination. Measurements of electron transport and fluorescence kinetics have confirmed that photosynthetic electron transport capacity is limited on the water splitting side of photosystem 2 in dark-grown seedlings, whereas the primary and secondary electron acceptors of photosystem 2 are fully synthesized and functioning in darkness. Polyethylene glycol must be used as a protective agent when isolating photoactive chloroplasts from secondary needles of conifers. However, the presence of polyethylene glycol, when isolating chloroplasts from dark-grown pine cotyledons, caused a total inhibition of the activity of photosystem 2. The failure of others to show a substantial electron transport activity in chloroplasts from dark-grown Pinus silvestris might depend on their use of polyethylene glycol in the preparation medium and/or on their use of suboptimal reaction conditions for the electron transport measurements.     

Growth Regulators and Wood Formation in Pinus silvestris

The formation of new xylem in the spring is preceded by bud development. In decapitated pine stem the formation of xylem is arrested until the outgrowth of interfascicular buds takes place. When indole-3yl-acetic acid (IAA) is applied to the cut surfaces of decapitated stems it induces the formation of a xylem ring on the whole length of 5-ycar old trees. Naphthaleneacetic acid (NAA) causes the formation of xylem; however, the width of the growth ring is several times broader at the point of application than at the base of the leader. Cis- and trans-cinnamic acids, coumarin, L-tryptophan, kinetin (Kin), benzylaminopurine (BAP) and gibberellic acid (GA) alone do not induce cambial divisions; however, GA and the cytokinins given jointly with IAA or NAA accelerated the basipetal stimulus which has been induced by the auxins, resulting in normal xylem formation. 2,3,5-Triiodobonzoic acid (TIBA) given jointly with IAA-induced formation of compression wood in the apical part of the stem and narrow diameter tracheids at the base. When carboxyl labelled IAA or NAA are applied to pine segments it is found that the basipetal movement of IAA is much quicker than that of NAA. GA and the cytokinins increase the rate of transport of both auxins, whereas TIBA arrests the bulk of auxin in the apical part of the stem.     

Studies on Sugars and Starch in Light- and Dark-Germinated Seeds of Scots Pine (Pinus silvestris)

Stachyose, raffinose and sucrose have been tentatively identified in ungerminated Scots pine seeds with paper chromatography and TLC. After the onset of the germination process even glucose and fructose were detected. No free galactose has been found. The occurrence and distribution of starch has been investigated with histochemical technique. The same substances have been quantitatively studied during the first 24 hours of the germination in light and darkness. This was done with an enzymatic technique alone or in combination with TLC. The evidence of a light influence on the transformations of starch, glucose and fructose has been discussed in relation to the phytochrome mechanism.     

Structural and functional relationships in developing Pinus silvestris chloroplasts

The light dependent chloroplast development of dark grown seedlings of Pinus silvestris L. was followed by analyses of chlorophyll content, chlorophyll a/b ratios, chlorophyll/P700 ratios, chlorophyll-protein complexes and structural changes. Low-temperature fluorescence emission spectra of isolated chloroplasts and separation of sodium dodecyl sulphate solubilized chlorophyll-protein complexes by gel electrophoresis showed that the chlorophyll-protein complexes of photosystem 1 (P700-CPa), photosystem II (PS II-CPa) and the light-harvesting complex LH–CPa/b were present in dark grown seedlings. The low-temperature fuoorescence emission maxima of isolated P700–CPa and PS II–CPa shifted towards longer wavelengths during greening in light, indicating a light induced change of the chlorophyll organisation in the two photosystems. Illumination caused LH–CPa/b to increase relative to P700–CPa, whereas the ratio between LH–CPa/b and PS II–CPa remained essentially constant. Analyses of low-temperature fluorescence spectra with or without 0.01 M Mg²⁺ showed that the Mg²⁺ controlled distribution of excitation energy into PS I was activated upon illumination of the seedlings. The photosynthetic unit size, as defined by the chlorophyll/P700 ratio, did not change over a 96 h illumination period, although the chlorophyll content increased about 6–fold during that time. This result and the constant electron transport rate per unit chlorophyll and time during chlorophyll accumulation provided evidence for a sequential development of the photosynthetic units when illuminating dark grown pine cotyledons. Electron micrographs showed that exposure of dark grown seedlings to light for 2 h caused the prolamellar body to disappear and grana to form. These changes occurred prior to substantial accumulation of chlorophyll or change in the ratio between LH–CPa/b and P700–CPa. However, both the water-splitting system of photosystem II and the Mg²⁺ controlled redistribution of excitation energy was activated during this period.     

Growth in Scots pine (Pinus silvestris L.)

Summary A theory of mechanisms underlying growth response to nitrogen in Scots pine is developed. Nitrogen in needles is considered as distributed in two mutually exclusive pools, viz. mobile and structurally bound. The size of the former pool, relative to total needle biomass, determines the rate of production of new needle biomass; this process decreases the pool size due to immobilization of mobile nitrogen. Some implications of the theory are derived and compared with published experimental results. It predicts a one-year time delay between a pulse fertilization and commencement of growth response, the latter having a total duration of about ten years with a maximum after about three; these predictions are not contradicted by available data.     

Seasonal variation in ribulose bisphosphate carboxylase activity in Pinus silvestris

Ribulose bisphosphate carboxylase activity was examined in Pinus silvestris L. during successive seasons. The enzyme activities were studied both in seedlings, kept under controlled conditions in a climate chamber, and in needles from a 15-year-old tree in a natural stand. The enzyme activities were analysed in cell-free extracts prepared with Tween 80 as protective agent. The carboxylase activity fluctuated periodically both in the seedlings and in the natural stand. In the seedlings, the weight-related activity in the older needles increased 50–100% (in the cotyledons c. 200%) in the beginning of the “summer”. It decreased as the new shoot developed. The specific activity increased c. 100%. With chlorophyll as base, the activity usually decreased during “summer”. In the developing current needles the carboxylase activity increased when expressed on a weight or on a protein basis. The decrease in weight-related carboxylase activity in the older needles was preceded by, or simultaneous with, loss of total protein. It is suggested that protein, including the carboxylase, is utilized as nitrogen reserve for the new shoot. During hardening by combined photoperiod and thermoperiod, the carboxylase activity decreased when expressed relative to dry weight and protein. Calculated on a chlorophyll basis, the activity was rather constant. In the natural stand the activity in the one- and two-year-old needles increased during spring and summer and decreased during autumn and winter. Even at severe winter stress substantial carboxylase activity remained in the needles. The activity of the enzyme in vivo is discussed with respect to electron transport and net photosynthesis.     

Studies on the Fat Metabolism of Light- and Dark-Germinated Seeds of Scots Pine (Pinus silvestris L.)

The aim of the present investigation has been to study whether the light factor influences the fat metabolism in the scots pine seeds in any way which could be correlated with its effect on the germination. The studies have been made on petroleum ether extracts. Gravimetric determinations showed small increases of extractable material during the first 24 hours of the germination process. After a longer time there was a small but insignificant consumption (72 hours). No effects of the light factor could be found. Investigations with gas-liquid chromatography on the corresponding extracts did not reveal any influence on the fatty acid composition, either of the light factor or of the germination period. The main identified fatty acids were linoleic, oleic, palmitic and stearic acid. One of the unidentified components (“c”) was also present in a large amount. Principally the same acids were found as free fatty acids but in different proportions: oleic, palmitic, linoleic and stearic acid. After the onset of the imbibition the linoleic acid appeared in the largest proportion. The total content of free fatty acids increased with increasing germination period. This was found both in titration experiments and in gas-liquid chromatography with an internal standard. The latter method permitted studies on the content of the individual acids. During the period 0–21 hours there were no evident differences between the content in light- and dark-germinated seeds, during the period up till 48 hours there was lower content in the light-germinated seeds concomitant with the onset of the mitotic activity in these seeds and after 48 hours there was a rapid increase in the same ones corresponding to the opening of the seed coats and the incipient protrusion of the rootlets. Thus changes of free fatty acid content were found only after the start of the light-induced growth but not under the preliminary phase, which earlier has been shown to be the determinative one in the light control of the germination.     

Two glycosides of a new dilignol from Pinus silvestris

The 4′-O-β-d-glucopyranoside and the 4′-O-α-l-rhamnopyranoside of 2,3-dihydro-7-hydroxy-2-(4′-hydroxy-3′- methoxyphenyl)-3-hydroxymethyl-5-benzofuranpropanol have been isolated and identified. Also isolated were two d-glucosides and an l-arabinoside of (+)-isolariciresinol and a l-rhamnoside, a d-xyloside and a d-glucoside of 1-(4-hydroxy-3-methoxyphenyl)- 2-[4-(3-hydroxypropyl)-2-hydroxyphenoxy]-1,3-propanediol.     

Occurrence of Indole-3-Acetic Acid in Buds of Pinus silvestris

The major ether-soluble, growth-stimulating substance detected by the Avena coleoptile straight-growth test in extract from sprouting buds of Scots pine (Pinus silvestris L.) was identified as indole-3-acetic acid by Rf values in 5 solvent systems and by its elution volume in ethanol on a Sephadex LH-20 column. When the substance was applied to the growth solution of wheat roots in a special test the growth in length of the roots was at first inhibited, but growth was recovered after about 6 hours in the same manner as when small quantities of IAA were applied. The extracts also contained large amounts of growth inhibitors which interfered with the auxin response if they were not removed.     

Frost hardiness development in Pinus silvestris seedlings in response to fertilization

The effects of different levels of nitrogen on freezing survival, the amounts of chlorophylls and β-carotene, the ratios Chl a/Chl b, and (Chl a + Chl b)/β-carotene in hardened and unhardened seedlings of Scots Pine (Pinus silvestris L.) have been investigated. There was. no correlation between frost hardiness and nitrogen content in the unhardened seedlings. However, the hardy seedlings with the highest nitrogen content, showed a decrease in frost hardiness of 3°C. The amounts of chlorophylls and β-carotene increased for all the treatments during hardening, except for the seedlings that had the highest nitrogen content; so that the effect of fertilizer was more evident in non-hardened than in hardened plants. The ratios Chl a/Chl b decreased during hardening in all the treatments. This was an effect of a larger increase of Chl b than Chl a. The ratios of Chl a + Chl b/β-carotene also decreased during hardening except for the plants that were fertilized with the second lowest amount of nitrogen. Since the amounts of Chl a and Chl b increase during hardening the amount of β-carotene increases even more.
The growth rates of the seedlings were closely related to the nitrogen content for the different treatments. All plants were infected with ectotrophic mycorrhizae, which resulted in an enhancement of ion transportation from the soil to the plants. This is important when the supply of ions is low.     

Mineral Nutrient Requirements of Pinus silvestris and Picea abies Seedlings

The mineral nutrient requirements of Pinus silvestris L. and Picea abies Karst. were studied according to previously published methods applied to a series of various plant species. The optimum nutrient proportions are similar to those of Vaccinium, with a lower relative potassium requirement than birch and other broad-leaf species. Various ratios between ammonium and nitrate nitrogen were about equally efficient except for a minor growth reduction with pure nitrate, which gave a comparatively low nitrogen content and a high cation/nitrogen ratio. The rate of ammonium uptake was much higher than that of nitrate when both sources were supplied. The required total concentration in the nutrient solution for maximum growth is lower in pine than in spruce, but both fall within the low salt range. Both species, especially pine, are sensitive to high salt concentrations. Although pine and spruce grow on the same type of soils as Vaccinium— leached soils with low base saturation – accumulation of calcium or other cations is not as pronounced as in Vaccinium, especially not in pine. The results are compared with results from similar experiments with a series of other conifers. All the conifers have more flexible cation uptake mechanisms than Vaccinium but the results indicate tendencies to accumulation of anions, nitrate and phosphate. Recommended fertilizer compositions for forest fertilization and nurseries are discussed.