The effect of sucrose on the differentiation of excised fern leaf tissue into either gametophytes of sporophytes

Excised juvenile leaves of Microgramma vacciniifolia (Polypodiaceae) develop sporophytic regenerants when grown on mineral agar with sucrose. The ratio of sporophytes to gametophytes produced from the leaf tissue increases with higher percentages of sucrose such that at 4% sucrose, the induction of aposporous gametophytes is a rare occurrence. Experiments varying the osmotic potential with sorbitol and those holding the osmotic potential of the culture medium constant while varying the sucrose level indicate that the effect of sucrose on the differentiation of fern leaf tissue into either gametophyte or sporophyte is nutritional rather than osmotic. A significant effect of sucrose in altering the differentiation of fern leaf tissue is the increased rate of senescence promoted by high sucrose concentrations.     

Comparative investigations on the distribution of sucrose synthase activity and invertase activity within growing, mature and old leaves of some C3 and C4 plant species

Sucrose synthase (EC 2.4.1.13) activity in young growing leaves was highest in the leaf base in eggplants ( Solanum melongena L.), cassava ( Manihot esculenta Crantz), grapevine ( Vitis vinifera L.), and in the leaf sheath of sugar cane ( Saccharum of ficinarum L.) and maize ( Zea mays L.). In addition, increasing sucrose synthase activity was measured towards the edge of growing eggplant leaves while the activity in mature leaves was highest in the midrib. The activity of acid and alkaline invertase was very low in the midrib but higher in the blade of fully expanded eggplant leaves. Highest invertase activities were found in younger growing leaves. It was concluded that in growing leaves a close relationship might exist between the activity of sucrose synthase and the import of sucrose from source leaves.
Detachment of mature eggplant leaves led to a 2- to 3-fold increase of sucrose synthase activity in blade and midrib of these leaves. In contrast, invertase activity decreased after detachment in both leaf blade and midrib. It was concluded that the rise in sucrose synthase activity might have been caused by the observed increase of sucrose concentration in detached leaves and that sucrose synthase might have an important role in the regulation of sucrose content of the conducting tissue.     

Apospory in leaf culture of staghorn fern (Platycerium bifurcatum)

The induction, origin, morphology, and ploidy of aposporous gametophytes produced on juvenile leaves of the fern Platycerium bifurcatum (Cav.) C. Chr. were studied. Leaf explants were grown on modified Murashige and Skoog medium with 0%, 0.01%, 0.1%, 1%, or 2% sucrose. A low sucrose concentration (0.01%) and wounding of the adaxial side of the leaf significantly increased the induction of aposporous gametophytes (90% of leaves produced gametophytes). Regeneration began as a proliferation of mainly epidermal cells on both sides of the leaf; subsequent development was similar to that shown by gametophytes originating from spores. Flow cytometric analysis of sporophytes and aposporous gametophytes revealed that both forms had the same ploidy level. On the basis of these findings, we propose a set of conditions which regularly and reproducibly induces apospory on most of the leaf explants of the fern P. bifurcatum.     

Comparison of sucrose metabolism during the rehydration of desiccation-tolerant and desiccation-sensitve leaf material of Sporobolus stapfianus

Sucrose accumulated during dehydration is a major potential energy source for metabolic activity during rehydration. The objective of the present study was to investigate aspects of leaf sucrose metabolism during the rehydration of desiccation-tolerant Sporobolus stapfianus Gandoger (Poaceae) over a 10-day period. Comparison was then made to sucrose metabolism during the rehydration of both desiccation-tolerant excised leaf material (dehydrated attached to the parent plant) and desiccation-sensitive leaf material (dehydrated detached from the parent plant to prevent the induction of tolerance) over a 48-h period. The pattern of sugar mobilization and glycolytic enzyme activity during the rehydration of the desiccation-tolerant excised leaves was similar to that in leaves attached to the parent plants. Significant breakdown of sucrose was not apparent in the initial phase of rehydration, suggesting the utilization of alternate substrates for respiratory activity. The desiccation-tolerant excised tissues provided a suitable control to compare the metabolism of rehydrating desiccation-sensitive material. In contrast to the tolerant tissues, sucrose breakdown in the sensitive leaves commenced immediately after watering and the accumulation in hexose sugars was inversely proportionate to the decrease in sucrose content. Hexokinase (EC 2.7.1.1), PFK (ATP phosphofructokinase, EC 2.1.7.11), aldolase (EC 4.1.2.13), enolase (EC 4.2.1.11), and PK (pyruvate kinase, EC 2.7.1.40) activity levels were significantly lower in the desiccation-sensitive material during rehydration.     

The effect of sucrose on the rate of de novo sucrose biosynthesis in leaf protoplasts from spinach, wheat and barley

Protoplasts from the leaves of wheat, spinach, and barley were found to synthesize [14C]sucrose from 14CO2 at rates comparable with those of the parent tissue. CO2 fixation and sucrose biosynthesis ceased virtually immediately when the light was switched off. The effect of sucrose pretreatment on the rate of de novo sucrose biosynthesis was found to vary with leaf age and with plant species. Protoplasts from young wheat and spinach leaves showed an apparent stimulation of the rate of sucrose biosynthesis after sucrose pretreatment. In protoplasts from mature leaves of spinach, sucrose pretreatment produced inhibition. After sucrose pretreatment protoplasts from mature spinach leaves showed low rates of CO2 fixation, and sucrose biosynthesis compared with controls. Conversely, with protoplasts from mature leaves of wheat and barley, the rate of CO2 fixation was unchanged and there was little or no effect on the rate of sucrose biosynthesis after sucrose pretreatment. Preincubation with sucrose had no effect on the activity of sucrose-phosphate synthetase (EC 2.4.1.14), cytoplasmic fructose-1,6-bisphosphatase (EC 3.1.3.11), or UDPglucose pyrophosphorylase (EC 2.7.7.9) from spinach leaves. It was concluded that there is no direct feedback inhibition of sucrose on the sucrose biosynthetic pathway in leaves of spinach, wheat, and barley. The mechanism of inhibition of sucrose biosynthesis by sucrose in spinach remains to be elucidated.     

Plant Vascular Architecture Determines the Pattern of Herbivore-Induced Systemic Responses in Arabidopsis thaliana

The induction of systemic responses in plants is associated with the connectivity between damaged and undamaged leaves, as determined by vascular architecture. Despite the widespread appreciation for studying variation in induced plant defense, few studies have characterized spatial variability of induction in the model species, Arabidopsis thaliana. Here we show that plant architecture generates fine scale spatial variation in the systemic induction of invertase and phenolic compounds. We examined whether the arrangement of leaves along the stem (phyllotaxy) produces predictable spatial patterns of cell-wall bound and soluble invertase activities, and downstream phenolic accumulation following feeding by the dietary specialist herbivore, Pieris rapae and the generalist, Spodoptera exigua. Responses were measured in leaves within and outside of the damaged orthostichy (leaves sharing direct vascular connections), and compared to those from plants where source-sink transport was disrupted by source leaf removal and by an insertional mutation in a sucrose transporter gene (suc2-1). Following herbivore damage to a single, middle-aged leaf, induction of cell-wall and soluble invertase was most pronounced in young and old leaves within the damaged orthostichy. The pattern of accumulation of phenolics was also predicted by these vascular connections and was, in part, dependent on the presence of source leaves and intact sucrose transporter function. Induction also occurred in leaves outside of the damaged orthostichy, suggesting that mechanisms may exist to overcome vascular constraints in this system. Our results demonstrate that systemic responses vary widely according to orthostichy, are often herbivore-specific, and partially rely on transport between source and sink leaves. We also provide evidence that patterns of induction are more integrated in A. thaliana than previously described. This work highlights the importance of plant vascular architecture in determining patterns of systemic induction, which is likely to be ecologically important to insect herbivores and plant pathogens.     

Effect of cutting on solute uptake by plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves.

The uptake of sucrose, 3-O-methylglucose (3-O-MeG), and valine were studied in discs and in purified plasma membrane vesicles (PMV) prepared from sugar beet (Beta vulgaris L.) exporting leaves. The uptake capacities of freshly excised leaf discs were compared with the uptake in discs that had been floated for 12 h on a simple medium (aging) and with discs excised from leaves that had been cut from the plant 12 h before the experiments (cutting). After cutting, sucrose uptake amounted to twice the uptake measured in fresh discs, whereas the uptake of 3-O-MeG and valine remained unaffected. In aged leaf discs, there was a general stimulation of uptake, which represented 400, 300, and 400% of the uptake measured in fresh discs for sucrose, 3-O-MeG, and valine, respectively. Sucrose uptake in fresh discs was sensitive to N-ethylmaleimide (NEM), to p-chloromercuribenzenesulfonic acid (PCMBS), and to mersalyl acid (MA). Although cutting induced the appearance of a sucrose uptake system that is poorly sensitive to NEM but sensitive to PCMBS and MA, aging induced the development of an uptake system that is sensitive to NEM but poorly sensitive to PCMBS and MA. Autoradiographs of discs fed with [14C]sucrose show that cutting resulted in an increase of vein labeling with little effect in the mesophyll, whereas aging induced an increase of labeling located mainly in the mesophyll. The data show that cutting is sufficient to induce dramatic and selective changes in the uptake properties of leaf tissues and that the effects of cutting and aging on the uptake of organic solutes are clearly different. Parallel experiments were run with purified PMV prepared from fresh and cut leaves. The uptake of sugars and amino acids was studied after imposition of an artificial proton motive force (pmf). Comparison of the uptake properties of PMV and of leaf tissues indicate that the recovery of the sucrose uptake system in PMV is better than the recovery of the hexose and of the valine uptake systems. As observed with the leaf discs, cutting induced a 2-fold increase of the initial rate of sucrose uptake in PMV but did not affect the uptake of valine and 3-O-MeG. Cutting induced an increase of both Vmax and Km of the sucrose transport system in PMV. Measurements of the pmf imposed on the vesicles indicated that the increase of sucrose uptake induced by cutting was not due to a better integrity of the vesicles. Hexoses did not compete with sucrose for uptake in PMV from fresh and cut leaves, and maltose was a stronger inhibitor of sucrose uptake in PMV from cut leaves than in PMV from fresh leaves. The sensitivity of sucrose uptake to NEM, PCMBS, and MA in PMV from fresh and cut leaves paralleled that described above for the corresponding leaf discs. These data show that (a) the changes induced by cutting on sucrose uptake by leaf discs are due to membrane phenomena and not to the metabolism of sucrose; (b) the study of sucrose uptake with PMB gives a good account of the physiological situation; and (c) the specific effects induced by cutting on the sucrose uptake system are not lost during the preparation of the PMV.     

The activity of uridine diphosphate glucose-d-fructose 6-phosphate 2-glucosyltransferase in leaves

1. By using EDTA in reaction mixtures it was possible to determine the activity of sucrose phosphate synthetase in freshly prepared leaf extracts without the complications caused by sucrose phosphatase. 2. EDTA was found also to increase the activity of sucrose phosphate synthetase by as much as 100%. 3. High sucrose phosphate synthetase activities were found in leaf preparations in which sucrose phosphatase was inhibited by EDTA. By contrast with previous reports, the activities were sufficient to allow sucrose synthesis in leaves during photosynthesis to occur via sucrose phosphate. 4. Sugar-cane plants having different rates of photosynthesis also had different activities of sucrose phosphate synthetase in their leaves. 5. It is suggested that the activity of sucrose phosphate synthetase in leaves may play a role in the control of the rate of photosynthesis.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

Plasma membrane vesicles from source and sink leaves : changes in solute transport and polypeptide composition

Plasma membrane vesicles (PMVs) were prepared by phase partitioning from microsomal fractions of either sink or source leaves of sugar beet (Beta vulgaris L.). The purity, the internal volume, the sidedness, and the sealingness of PMVs prepared from sink leaves did not differ from those measured with PMVs from source leaves. Yet, in response to an imposed proton motive force, PMVs from source leaves accumulated about 4-fold more sucrose than PMVs from sink leaves. The developmental stage did not affect the uptake of glucose and valine in PMVs prepared from leaf tissues. It was concluded that the sink/source transition is accompanied either by the incorporation into the plasma membrane of leaf cells of proteins mediating proton-sucrose cotransport, or by their activation. N-ethylmaleimide and a polyclonal ascitic fluid directed against the 42-kD region of the plasma membrane containing a putative sucrose carrier inhibited the uptake of sucrose in PMVs from source leaves, but not in PMVs from sink leaves. Sodium dodecyl sulfate gel electrophoresis and western blot suggested that the 42 polypeptide was more abundant in the PMVs from source leaves than in the PMVs from sink leaves.     

Sucrose-Induced Expression of Genes Coding for the Tuberous Root Storage Protein, Sporamin, of Sweet Potato in Leaves and Petioles

Sporamin, a major tuberous root protein of sweet potato, wasfound to accumulate in large quantities in excised leaves andpetioles when such explants were supplied with high concentrationsof sucrose. Although a small amount of sporamin could be detectedin leaves and petioles treated with 1% or lower concentrationsof sucrose, the maximum level of induction required sucroseat a concentration of 3% or higher. The appearance of sporaminpolypeptides in leaves and petioles treated with 3% sucrosefollowed a lag period of about one day, while a significantamount of sporamin mRNAs was already detectable in petiolesafter one day of treatment with sucrose. Addition of silvernitrate to the medium did not affect the accumulation of sporamin,suggesting that this induction is not due to the effect of ethyleneinduced by wounding of the tissue. The accumulation of sporamincould also be induced by glucose and by fructose, but not byman-nitol, suggesting that changes in carbohydrate and/or energymetabolism in the cell may be involved in the induction. Callustissues obtained by treatment of leaf segments with 1-naphthaleneaceticacid did not accumulate sporamin even though these cells werecultured on agar medium that contained 3% sucrose. However,when callus tissues were allowed to grow after transfer to amedium that contained 6-benzylaminopurine and sucrose, accumulationof large amounts of sporamin was induced. These results suggestthat, while expression of genes coding for sporamin can be inducedin organs other than the tuberous root by a process that doesnot accompany the differentiation of tissue, the induction ofexpression of sporamin genes by sucrose requires that cellsbe competent in some specific, but as yet unidentified, way. (Received August 27, 1990; Accepted November 5, 1990)     

Comparative studies of sucrose and mannitol utilization in celery (Apium graveolens)

Utilization of sucrose and mannitol, the major forms of translocatable assimilate in celery ( Apium graveolens L. cv. Giant Pascal), was investigated in intact plants, excised leaves and leaf discs by estimating the soluble carbohydrate pools, starch levels and oxidation of [¹⁴C]-sucrose or mannitol in the light and after extended dark treatments. In detached mature fully-expanded leaves, mannitol pools remained constant, while sucrose decreased during a 48 h dark treatment. In attached leaves on plants trimmed to a single compound leaf, however, mannitol levels decreased after a dark treatment. In leaf discs floated on bathing solutions containing [¹⁴C]-sucrose or [¹⁴C]-mannitol, oxidation of mannitol was restricted to young leaf tissues, whereas sucrose was metabolized to CO₂ regardless of leaf age. Uptake of labelled mannitol, however, was greater than that of sucrose in the light in leaves of every age. Although both mannitol and sucrose are translocated out of leaf tissues, leaf age differences indicate that, unlike sucrose, mannitol utilization is restricted to active sink tissues. The results suggest different roles for mannitol and sucrose with mannitol representing a more rigorously sequestered transport carbohydrate.     

Comparison of the effect of rapidly and gradually developing water-stress on carbohydrate metabolism in spinach leaves

Abstract. The effect of water-stress on photosynthetic carbon metabolism in spinach ( Spinacia oleracea L.) has been studied in experiments in which water-stress was induced rapidly by floating leaf discs on sorbitol solutions or wilting detached leaves, and in experiments in which water-stress was allowed to develop gradually in whole plants as the soil dried out. In both short- and long-term water stress, the rate of photosynthesis in saturating CO₂ did not decrease until leaf water potential decreased below -1.0 MPa. However, at smaller water deficits there was already an inhibition of starch synthesis, while sucrose synthesis remained constant or increased. This change in partitioning was accompanied by an increase in activation of sucrose-phosphate synthase (revealed as an increase in activity assayed in the presence of low hexose-phosphate and inorganic phosphate, while the activity assayed with saturating hexosephosphates remained unaltered). Water-stressed leaves had a two- to three-fold higher sucrose content at the end of the night, and contained less starch than non-stressed leaves. When leaves were held in the dark, sucrose was mobilized initially, while starch was not mobilized until the sucrose had decreased to a low level; in water-stressed leaves, starch mobilization commenced at a two-fold higher sucrose content. It is concluded that water-stressed leaves maintain higher sucrose and lower starch levels than non-stressed leaves. This response is found in rapid and long-term stress, and represents an inherent response to water deficits.     

Biosynthesis of Sucrose and Mannitol as a Function of Leaf Age in Celery (Apium graveolens L.)

In celery (Apium graveolens L.), the two major translocated carbohydrates are sucrose and the acyclic polyol mannitol. Their metabolism, however, is different and their specific functions are uncertain. To compare their roles in carbon partitioning and sink-source transitions, developmental changes in ¹⁴CO₂ labeling, pool sizes, and key enzyme activities in leaf tissues were examined. The proportion of label in mannitol increased dramatically with leaf maturation whereas that in sucrose remained fairly constant. Mannitol content, however, was high in all leaves and sucrose content increased as leaves developed. Activities of mannose-6-P reductase, cytoplasmic and chloroplastic fructose-1,6-bisphosphatases, sucrose phosphate synthase, and sucrose synthase increased with leaf maturation and decreased as leaves senesced. Ribulose bisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehydrogenase activities rose as leaves developed but did not decrease. Thus, sucrose is produced in all photosynthetically active leaves whereas mannitol is synthesized primarily in mature leaves and stored in all leaves. Onset of sucrose export in celery may result from sucrose accumulation in expanding leaves, but mannitol export is clearly unrelated to mannitol concentration. Mannitol export, however, appears to coincide with increased mannitol biosynthesis. Although mannitol and sucrose arise from a common precursor in celery, subsequent metabolism and transport must be regulated separately.     

Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis

It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K_m [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought.     

Source and sink leaf metabolism in relation to Phloem translocation: carbon partitioning and enzymology

The import-export transition in sugar beet leaves (Beta vulgaris) occurred at 40 to 50% leaf expansion and was characterized by loss in assimilate import and increase in photosynthesis. The metabolism and partitioning of assimilated and translocated C were determined during leaf development and related to the translocation status of the leaf. The import stage was characterized by C derived from either ¹⁴C-translocate or ¹⁴C-photosynthate being incorporated into protein and structural carbohydrates. Marked changes in the C partitioning were temporally correlated with the import-export conversion. Exporting leaves did not hydrolyze accumulated sucrose and the C derived from CO₂ fixation was preferentially incorporated into sucrose. Both source and sink leaves contained similar levels of acid invertase and sucrose synthetase activities (sucrose hydrolysis) while sucrose phosphate synthetase (sucrose synthesis) was detected only in exporting leaves. The results are discussed in terms of intracellular compartmentation of sucrose and sucrose-metabolizing enzymes in source and sink leaves.     

Does Apoplastic Sucrose Induce the Ability for Sucrose Loading in Developing Leaves of Sugarbeet?

Dark-grown sugarbeet (Beta vulgaris L.) leaves were used toinvestigate a possible role of apoplastic sucrose in the inductionand development of the putative phloem-located sucrose carrierin relation to minor vein loading and export capacity. Unlabeledsucrose was introduced to the leaf apoplast after which veinaccumulation of [¹⁴C]sucrose was determined by autoradiogra-phy.Western blotting was used to detect the putative carrier. Anaffinity purified antibody against the sucrose binding proteinof soybean did not cross-react with the protein in a plasmalemma-enrichedfraction from sugarbeet leaves. Challenging the apoplast ofleaf discs with buffer plus sucrose for 6 h (induction) resultedin decreased [¹⁴C]sucrose uptake. When induction treatmentswere conducted with detached intact leaves in the dark, sucroseand glucose, but not buffer alone enhanced [¹⁴C]sucrose uptake.Detached leaves induced under laboratory light conditions for24 h showed enhanced [¹⁴C]sucrose uptake even in the absenceof any sugar introduced to the apoplast (buffer only). The datasuggested that in the etiolated tissue, sucrose was not a directand specific inducer of its putative carrier; instead sugarsmay have provided the energy for vein loading. Furthermore,the data suggested a role for light in the development of theputative sucrose carrier and vein accumulation of sucrose intransitional leaves of sugarbeet. The role of light may alsobe related to tissue energy level. ¹Contribution No. D-15192-1-91 from the New Jersey AgriculturalExperiment Station. This work was funded in part by the BeetSugar Development Foundation and Rutgers University ResearchCouncil and was submitted as partial fulfillment for M.S. degreeby Lynne H. Pitcher. (Received February 19, 1991; Accepted May 13, 1991)     

三种柚子品种的离体快速繁殖

以三种柚子（Citrus grandis L.）品种叶片为外植体诱导愈伤组织,以幼嫩茎段诱导不定芽和不定根。结果表明：1.0 mg/L2,4-D与6%蔗糖处理的叶片愈伤组织诱导率最高,最高诱导率达82.5%;6-BA0.5 mg/L和NAA0～0.5 mg/L激素配比诱导丛生芽效果最高,最高可在每个茎段上诱导4个不定芽;1/4 MS＋IBA 2.0 mg/L诱导生根效果最好,最高达40%,虽然根系很弱,难以成苗,但可以用于试管微嫁接。所有的培养中,？溪蜜柚和沙田柚的诱导率接近,都比江津柚高大约30%～50%。