14C-Studies on Apple Trees. I. The Effect of the Fruit on the Translocation and Distribution of Photosynthates

The presence of fruits affects the translocation and distribution of photosynthates from apple leaves to other organs of the tree. An attempt has been made to study the relationship in greater detail by following the distribution of ¹⁴C introduced in the form of ¹⁴CO₂ on shoots with and without fruits, respectively. Determinations of the ¹⁴C-content were made on different parts of the shoot sampled at varying intervals after the introduction of the tracer. The ^l4C-labelling and the content of sorbitol and sugar in the leaves were determined by means of paper chromatography. A total of nearly 90 per cent of the ¹⁴C taken up by the leaves can be transferred to a fruit close by, the majority during the first 4 to 5 days following the addition of the ¹⁴C. The content of ¹⁴C in the leaves is reduced more rapidly in shoots with fruits than in those without. Young leaves retain more of the added ¹⁴C than do fully developed ones. The greatest changes with time are found in the methanol-soluble ¹⁴C-compounds. Immediately following application, the leaves contain 58 to 80 per cent of the ¹⁴C added in sorbitol, 7 to 9 per cent in sucrose, and 1 to 4 per cent in the form of glucose. Within 5 days after the introduction of ¹⁴C the amount of ¹⁴C-sorbitol is reduced very considerably, while in certain cases the amount of ¹⁴C-glucose increases. The ¹⁴C-sorbitol content was higher in leaves from shoots without fruits than in those from fruit-bearing shoots, and this applied also to the total contents of sorbitol and of glucose.     

14C-Studies on Apple Trees. V. Translocation of Labelled Compounds from Leaves to Fruit and their Conversion within the Fruit

Following application of ¹⁴CO₂ to fruit spur leaves, the majority of the ¹⁴C absorbed is transfered to the fruit on the same spur, and the total content of ¹⁴C within the leaf-fruit system as a whole remains virtually constant with time. The considerable reduction in activity in the leaves is accounted for mainly by a decrease in the amount of ¹⁴C-sorbitol, although relatively speaking the decrease in ¹⁴C-sucrose is also considerable. The major part of the activity of the sugar fraction in the conducting tissues between blade and fruit (petiol, spur) is found in sorbitol. Immediately following uptake of ¹⁴C yia the leaves a large part of the activity of the sugar fraction in the fruit is found in sorbitol; but this activity is rapidly reduced, accompanied by an increase in sucrose activity, and over longer periods of time increases in particular in glucose and fruclose activity, and in that of methanol insoluble compounds. The changes in activity distribution in the fruit vary with the variety of fruit and the dates within the growing season. By injecting labelled sorbitol directly into the fruit sorbitol is converted into sucrose, glucose and fructose, while injection of labelled sucrose, glucose and fructose has yielded proof of interconversions between these compounds but no measurable amounts of surbitol. After application of ¹⁴CO₂ directly to the outer skin of the fruit considerably less of the activity is found in sorbitol than is the case in leaves following exposure to ¹⁴CO₂. A minor, but significant, translocation of ¹⁴C away from the fruit was found to take place following the application of labelled ¹⁴C compounds to the fruit. The smallness of the respiratory loss of ¹⁴C in the leaf-fruit system is discussed. It is concluded that in apple trees considerable translocation occurs in the form of sorbitol which in the fruits rapidly converted into other compounds.     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.     

The Export and Distribution of 14C-labelled Assimilates from each Leaf on the Shoot of Lolium temulentum during Reproductive and Vegetative Growth

In both reproductive and vegetative plants of Lolium temulentumL., the export of ¹⁴C-labelled assimilates from each healthyleaf on the main shoot to terminal meristem, stem, tillers,and roots was measured each time a new leaf was expanded, fora period of 5 to 6 weeks. Some labelled assimilates moved fromeach leaf on the main shoot to every meristem in the same shoot,as well as to the tops and roots of adjacent organically attachedtillers. The terminal meristem of the reproductive shoot, which includedthe developing inflorescence, received 70–80 per centof the carbon assimilated by the emerged portion of the growingleaf, 15–25 per cent of the carbon assimilated by thetwo youngest expanded leaves, and 5–10 per cent of thatfrom each of the older leaves. A similar pattern of carbon supplyto the terminal meristem was found in vegetative shoots, exceptthat older leaves on young vegetative shoots supplied even lessof their carbon to the terminal meristem. The general conclusionis that developing leaves at the tip of the shoot receive aboutthe same proportion of carbon from each leaf as does a developinginflorescence. Young expanded leaves provided most labelled assimilates forstem growth; during both reproductive and vegetative growth,expanded leaves increased their export of labelled carbon tostem, and exported less of their ¹⁴C to roots and sometimesto tillers. In these reproductive and vegetative shoots, grown in a constantexternal environment, the major changes in the pattern of distributionof labelled assimilates appeared to be the result of increasedmeristematic activity in stem internodes; the development ofan inflorescence had no obvious direct effect on the carboneconomy of shoots.     

Apoplastic Transport of 14C-Photosynthates Measured under Drought and Nitrogen Supply

Using water infiltration of the plant and individual shoots with the subsequent intercellular liquid extraction by the pressure chamber, dynamics of the movement ¹⁴C-photosynthates from cell to apoplast, and ¹⁴C distribution among photosynthetic products in mesophyll cells and apoplast were studied. The relative quantity of ¹⁴C-photosynthetes in leaf apoplast depended on growing conditions; drought increased, and nitrate supply decreased it. When the middle leaves absorbed ¹⁴CO₂, photosynthates moving down in stem phloem appeared in intercellular space, where they were transported up by transpiration stream. ¹⁴C-photosynthates entering to the apex and young leaves were utilized a accumulated, and photosynthates transported to the mature leaves were reloaded into the phloem and reexported. Thus, photosynthates circulated through the plant and were redistributed to the plant organs according to their transpiration. In leaf apoplast photosynthetic sucrose was partly hydrolyzed to glucose and fructose. This increased under high nitrogen supply. The result indicate that apoplast sucrose hydrolysis is the basic cause of the reduction of photosynthate flux from leaves when the nitrate concentration in soil increases.     

14C-studies on Apple Trees. IX. Seasonal Changes in the Formation of Fruit Constituents and their Subsequent Conversions

The formation and subsequent conversions of ¹⁴C-labelled compounds were followed in fruits of Malus domestica cvs. Golden Delicious and Cox's Orange Pippin after labelling proximate leaves with ¹⁴CO₂ at different times during the growing season. A few hours after labelling of the leaves, the larger share of fruit ¹⁴C was detected in sorbitol. This share descreased rapidly except in the late autumn. When labelling about 1 July (c. 1 month after bloom), 40–60% of the fruit ¹⁴C was permanently fixed in the methanol and water insoluble fraction. 25% or more was primarily found in organic acids, but this declined during the season to a few per cent. When labelling at the end of July, the dominating feature was the establishment of a peak of temporarily insoluble ¹⁴C, returning back to the soluble form through October and November. This was particularly pronounced in‘Cox's Organe Pippin'. Labelling with ¹⁴C at the end of August and at the end of September yielded increasing amounts of ¹⁴C in sugars. The labelling of fructose predominated, but as the autumn progressed the amount of label in sucrose increased. This was due to a conversion from ¹⁴C-compounds of older origin as well as to a larger share of the imported assimilates turning into sucrose at this time of the year. During prolonged storage of harvested fruits at 3°C, ¹⁴C in fructose increased at the expense of ¹⁴C in sucrose.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

Accumulation of 32P and [14C]sucrose in decapitated and intact shoots of the fern Davallia trichomanoides blume

The transport and accumulation of ³²P and [¹⁴C] sucrose in decapitated and intact shoot segments of the fern Davallia were studied. The apical buds of intact shoots and the expanding buds of shoots decapitated 4 weeks before application are major sinks for these nutrients. Decapitation results in a shift of ¹⁴C accumulation from the apex to the lateral buds within 36 h. This shift can be reversed in shoots decapitated for 12 h by replacing the apex. Increased ¹⁴C accumulation into the stump region occurs when decapitated shoot segments are treated with indole-3-acetic acid, and decreased label accumulation into the apical region results when intact shoots are treated with 2,3,5-triiodobenzoic acid.     

14C-Studies on Apple Trees III. The Influence of Season on Storage and Mobilization of Labelled Compounds

The present paper reports an attempt to elucidate the storage and mobilization processes in 1-year-old apple rootstocks by studying the ¹⁴C content of different parts of the tree following application of ¹⁴CO₂. The translocation of the ¹⁴C taken up from the leaves to other parts proceeds at the highest rate during the first few days after the application of ¹⁴C. The distribution in shoots, trunk and roots after application of ¹⁴C during May, July, August, and in part of September depends in particular upon the intensity of growth in the various parts. From the lime of the application of ¹⁴C until leaf-fall, 40–50 per cent of the ¹⁴C initially absorbed disappears from the tree. After exposure to ¹⁴C during October, and in part of September, a relatively large part of the ¹⁴C applied goes to the root. In this case there is a considerable reduction in the ¹⁴C-conlent from leaf-fall to the following spring after leafing, especially in the root, although relatively speaking reduction is also considerable in the bark of the parts above ground, and it is most pronounced in the methanol (80 %) soluble fraction. The reduction takes place primarily during spring, and comprises, after application during October, for the whole tree 20–25 per cent of the ¹⁴C initially absorbed. Only 13–17 per cent of this amount was recovered in the newly developed shoots and leaves in the following June.     

Shoot regeneration in response to carbon source on internodal explants of Annona muricata L.

Adventitious shoot regeneration from internodal explants of mature plants of Annona muricata L. was obtained on Nitsch media. Meristems were induced with sorbitol as the sole carbon source supplemented with 2 mg l^–1 of benzylaminopurine and 0.5 mg l^–1 naphthaleneacetic acid. Adventitious shoots were developed only when the explants were transferred onto growth regulator-free media containing sucrose, galactose, or glucose. A hypothesis is proposed for the involvement of sorbitol in the induction and development of de novo shoots from internodal explants of mature trees of A. muricata.     

14C-Studies on Apple Trees. VIII. The Seasonal Variation and Nature of Reserves

The nature, seasonal variation and mobilization of reserves in Malus × domestica have been studied by means of ¹⁴C, carbohydrate analyses and extractions of xylem sap. Following exposure to ¹⁴CO₂ in the autumn, the majority of the ¹⁴C absorbed is found in the root. During the winter and in particular the spring the amounts of ¹⁴C in the top and root are reduced to approximately 40 per cent of the autumn values; in the root the amount of dry matter was also considerably reduced. In the tops, most of the ¹⁴C absorbed was found as methanol (80 %)-soluble ¹⁴C which also showed the greatest seasonal reduction; sorbitol, sucrose or glucose in particular are responsible for the decrease in concentration within this fraction. Maximum values for methanol-insoluble ¹⁴C were found in March. In the root, the highest values for absolute changes were found for methanolinsoluble ¹⁴C. Hydrolysis of this fraction showed considerable activity in glucose. In the root there was also considerable activity in a precipitated fraction of the methanol extract. Eluates of xylem sap from apple branches contained primarily sorbitol, the highest concentration of which was found at the beginning of March. For a tree with a dry matter weight of about 300 g, the utilization of reserves from the tree in the spring was calculated to be at least 13 g of dry matter. However, only a minor part (less than 25 per cent) of the latter appears to serve as building material for new growth.     

Effects of carbon source and concentration on development of lingonberry (<Emphasis Type="Italic">Vaccinium vitis-idaea</Emphasis> L.) shoots cultivated <Emphasis Type="Italic">in vitro</Emphasis> from nodal explants

Summary Carbohydrate type and concentration and their interactive effects on in vitro shoot proliferation of three lingonberry (Vaccinium vitis-idaea ssp. vitis-idaea L.) cultivars (‘Regal’, ‘Splendor’, and ‘Erntedank’) and two V. vitis-idaea ssp. minus (Lodd) clones (‘NL1’ and ‘NL2’) were studied. Nodal explants were grown in vitro on medium with 2 μM zeatin and either glucose, sorbitol, or sucrose at a concentration of 0, 10, 20, or 30 gl⁻¹. The interactive effects of carbohydrate type and concentration and genotype were important for shoot proliferation. The best response was afforded by sucrose at 20 gl⁻¹ both in terms of explant response and shoot developing potential, although glucose supported shoot growth equally well, and in ‘NL1’ at 10 gl⁻¹ it resulted in better in vitro growth than sucrose. Carbohydrate concentration had little effect on shoot vigor. The genotypes differed in terms of shoots per explant, length, and vigor, leaves per shoot, and callus formation at the base of explants; this was manifested with various types and concentrations of carbohydrate. Changing the positioning of explants on the medium from vertically upright to horizontal increased the shoot and callus size, but decreased shoot height and leaves per shoot. Proliferated shoots were rooted on a peat:perlite (1∶1, v/v) medium and the plantlets were acclimatized and eventually established in the greenhouse.     

Distribution patterns of assimilated 14C in vegetative and reproductive shoots of Lolium perenne and L. temulentum

The movement of ¹⁴C-labelled assimilate to the terminal meristem, stem, mature leaves, tillers and roots was measured in Loliurn perenn and Lolium temulentum after exposure to ¹⁴C0₂ of the youngest fully-expanded leaf and, on fewer occasions, the oldest healthy leaf on the main shoot. During early vegetative growth, the terminal meristem, tillers and roots received most of the ¹⁴C exported from the youngest leaf. As the shoot aged, more ¹⁴C was exported to the terminal meristem and tillers and less to roots. When the stem became a sizeable sink for ¹⁴C at the six-leaf (L. temulentum) or eleven-leaf (L. perenne) stage, less ¹⁴C moved to tillers and much less to roots. The terminal meristem continued to receive ¹⁴ at a steady rate throughout late vegetative growth. The transition from vegetative to reproductive growth in both species was marked by an abrupt increase in the export of ¹⁴C to stem from the upper leaf, but there was little change in the proportion of ¹⁴C which moved to the developing leaves and incipient inflorescence at the terminal meristem. At the same time, less ¹⁴C moved to tillers and much less to roots. Immediately before ear emergence, the export of ¹⁴C from the upper leaf (flag leaf) to the stem declined and the proportion moving to the ear increased, reaching a maximum of 55–75% as the ear emerged. The relative patterns of export of upper and lower leaves showed that while some ¹⁴ moved from each leaf to all meristems, the proximity of actively growing meristems appeared to be the main factor which determined the destination of most exported ¹⁴C. The distribution of ¹⁴C from upper and lower leaves was most alike in young vegetative plants of L. perenne. At later stages of development of both species, the terminal meristem and stem received most 14¹⁴C from the upper leaf, while roots and tillers received mos 14¹⁴C from the oldest leaf at the base of the shoot.     

Carbon partitioning into sorbitol,sucrose, and starch in source and sink apple leaves as affected by elevated CO2

Experiments were conducted in controlled growth chambers to evaluate how increases in CO₂ concentration ([CO₂]) affected carbon metabolism and partitioning into sorbitol, sucrose, and starch in various ages of apple leaves. Apple plants (Malus domestica), 1 year old, were exposed to [CO₂] of 200, 360, 700, 1000, and 1600 μl l⁻¹ up to 8 days. Six groups of leaves (counted from the shoot apex): leaves 1–5 (sink), 6–7 (sink to source transition), 8–9 (sink to source transition), 10–11 (nearly-matured source), 21–22 (mid-age source), and 30–32 (aged source), were sampled at 1, 2, 4, and 8 days after [CO₂] treatments for carbohydrate analysis. Increases in [CO₂] from a sub-ambient (200 μl l⁻¹) to an ambient level (360 μl l⁻¹) significantly increased the concentrations of sorbitol, sucrose, glucose, and fructose tested in all ages of leaves. Continuous increase in [CO₂] from ambient to super-ambient levels up to 1600 μl l⁻¹ also increased sorbitol concentration by ≈50% in source leaves, but not in sink and sink to source transition leaves. Increases in [CO₂] from 360 to 1600 μl l⁻¹, however, had little effect on sucrose content in all ages of leaves. Starch concentrations increased in all ages of leaves as [CO₂] increased. Rapid starch increases (e.g. 5-, 6-, 20-, and 50-fold increases for leaf groups 1–5, 6–7, 10–11, and 21–22, respectively) occurred from 700 to 1600 μl l⁻¹ [CO₂] during which increases in sorbitol concentration either ceased or slowed down. Our results indicate that changes in carbohydrates were much more responsive to CO₂ enrichment in source leaves than in sink and sink to source transition leaves. Carbon partitioning was favored into starch and sorbitol over sucrose in all ages of leaves when [CO₂] was increased from 200 to 700 μl l⁻¹, and was favored into starch over sorbitol from 700 to 1600 μl l⁻¹ [CO₂].     

Sorbitol metabolism by apple seedlings

The aims of this work were to compare the roles of sorbitol and sucrose in seedlings of Malus domestica, to discover which tissues synthesize sorbitol and which break it down, and to examine these tissues for enzymes of sorbitol metabolism. The detailed distribution of label was determined after supplying intact seedlings with ¹⁴CO₂, and excised parts of seedlings with [U-¹⁴C]fructose and [U-¹⁴C]sorbitol. The results showed that appreciable synthesis of sorbitol occurred only in the leaves but did not depend directly on photosynthesis. All tissues examined metabolized sorbitol but metabolism was extensive only in root apices, and in leaves which had been kept in the dark. The above experiments suggest that sorbitol supplements but does not replace sucrose. Extracts of apple leaves showed no trace of either a polyol or a polyol phosphate dehydrogenase but did exhibit sorbitol-6-phosphate phosphatase activity. A limited number of experiments with extracts of the blades of Laminaria digitata indicated that they contained mannitol-1-phosphate phosphatase and mannitol dehydrogenase.     

Glucofructosan metabolism in Cichorium intybus roots

A 10-fold purification of sucrose sucrose fructosyl transferase from Cichorium intybus roots was achieved by ammonium sulphate fractionation and DEAE-cellulose column chromatography. The energy of activation for this enzyme was ca 48 kJ/mol sucrose. Sucrose sucrose fructosyl transferase and invertase were prominent during early months of growth. Evidence obtained from: (1) the changes in carbohydrate composition at monthly intervals; (2) comparative studies on fructosyl transferase and invertase at different stages of root growth; and (3) incubation studies with [¹⁴C]glucose, [¹⁴C]fructose and [¹⁴C]sucrose revealed that, during the later stages of root growth, fructosan hydrolase is responsible for fructosan hydrolysis. No evidence for the direct transfer of fructose from sucrose to high M_r glucofructosans was obtained.     

Patterns of 14C-labelled Assimilate Partitioning in Red and White Clover During Vegetative Growth

A quantitative analysis of the ¹⁴C-labelled assimilate suppliedby the expanded leaves on the primary shoot to growing leaves,stem, lateral shoots (branches or stolons) and roots in redand white clover was conducted during vegetative growth. Stem growth of the primary shoot was inhibited in both cloversand utilized no energy resources. The growing leaves at theprimary shoot apex of white clover imported 4 per cent of theshoot's assimilate compared with 10 per cent in red clover.At the basal end of the primary shoot, the tap root of whiteclover imported 16 per cent of the shoot's assimilate comparedwith 22 per cent in red clover. Branches in red clover and stolonsin white clover were by far the largest sinks for primary shootassimilate, importing 39 per cent and 63 per cent of the labelledassimilate, respectively. Analyses of the translocation of assimilate from individualprimary shoot leaves demonstrated that in both clovers olderleaves exported more of their assimilate to branches or stolons,whereas younger leaves exported more of their assimilate toroots, and possibly in white clover, to growing leaves at thetip of the shoot. Of the labelled assimilate exported to branchesor stolons, each primary shoot leaf exported preferentiallyto the branch or stolon in its own axil, but in addition exportedsubstantial quantities of assimilate to all other axillary shoots,particularly those arising from basal axils where the subtendingleaf had died. Trifolium repens, Trifolium pratense, red clover, white clover, assimilate partitioning, perennation     

Interconversions of 14C-labelled Sugars in Apple Tree Tissues12

Tissue pieces excised from orchard-grown apple trees duringa growing season exhibited different and changing capabilitiesof transferring ¹⁴C-label from sucrose, fructose and sorbitolto other soluble carbohydrates. All tissues incorporated fructose¹⁴C into sucrose but only leaves incorporated significant amountsof label from sucrose into sorbitol. As seeds developed andmatured, their ability to incorporate ¹⁴C from sorbitol andfrom fructose into sucrose increased. Sorbitol and sucrose arethe major translocated photosynthetic products of apple leavesbut whereas sorbitol appears to be an end-product of synthesis,sucrose may be considered as a substrate involved more directlyin carbohydrate utilization. Key words: Malus domestica, Apple, Carbohydrate interconversions     

14C-Studies on Apple Trees. VII. The Early Seasonal Growth in Leaves, Flowers and Shoots as Dependent upon Current Photosynthates and Existing Reserves

The young leaves' consumption proper of photosynthates and their contribution to the growth of flowers, fruits and shoots by exposing spurs and shoots to ¹⁴CO₂ at the earliest stages of the growth period in apple trees (Malus X domestica) were studied. By a parallel determination of the growth intensity in various organs an attempt is made to evaluate their relative dependence on current photosynthates and on reserves from inside the tree. The proper fixation of ¹⁴C by growth in the exposed leaves is high in the earliest phases of growth. The fixation of ¹⁴C is considerable in the flowers, including the petals, immediately prior to flowering, in intensely growing fruits, and in the woody parts of the current year's shoots, when the main part of the terminal growth has been completed. Under conditions of high intensity of growth in an organ, the total fixation by growth in the parts studied may amount to as much as 80–90% of the ¹⁴C absorbed. Only in the very earliest phases of development does the growth of flowers and shoots appear to be based to a greater extent on materials supplied from reserves than from current photosynthesis. Quantitatively the greater part by far of the total new growth in fruits and shoots appears to be based on materials from current photosynthesis.