Subcellular volumes and metabolite concentrations in spinach leaves

Cellular and subcellular volumes in mature leaves of spinach (Spinacia oleracea L. US Hybrid 424) were determined stereologically from light and electron micrographs. Forty-nine-day-old leaves of spinach with a total leaf volume of 1177 μL per mg chlorophyll (Chl) were found to be composed of 3% epidermis, 58% mesophyll, 1% vascular tissue, 5% apoplasm and 32% gas space. In the epidermal cells 89% of the volume was occupied by the vacuole. The mesophyll cells consisted, expressed in mg·Chl⁻¹, of 546 μL (79%) vacuole, 66 μL (9.5%) chloroplast stroma, 24 μL (34%) cytosol, 3.7 μL (0.5%) mitochondria and 2.1 μL (0.3%) nucleus. From previous measurements of the subcellular levels of sucrose, of phosphorylated intermediates of carbohydrate metabolism, of malate, oxoglutarate and various amino acids in illuminated leaves, and the above subcellular volumes, the corresponding subcellular metabolite concentrations have been determined. Of the substances measured, only with malate was the concentration higher in the vacuole than in the cytosol. The concentration of sucrose in the cytosol was 5 times, and that of amino acids even 30 times higher than in the vacuole.     

Subcellular Volumes and Metabolite Concentrations in Potato (Solanum tuberosum cv. Désirée) Leaves1

Cellular and subcellular volumes in mature leaves of potato (Solanum tuberosum cv. Désirée) were determined stereologically from light and electron micrographs. Leaves of ten-week-old plants with a total leaf volume of 623 μL per mg chlorophyll (Chl) were found to be composed of 12 % epidermis, 68 % mesophyll, 5 % vascular tissue, 3 % apoplast and 16 % gas space. In the epidermal cells 97 % of the volume was occupied by the vacuole. The mesophyll cells consisted (as expressed per mg Chl) of 323 μL (76 %) vacuole, 35 μL (8 %) chloroplast stroma, 22 μL (5 %) cytosol plus nucleus and peroxisomes, and 4μL, (1 %) mitochondria. A comparison of these values with subcellular volumes previously determined for spinach and barley leaves, shows that the relative sizes of the subcellular volumes are strikingly similar. Subcellular concentrations of carbohydrates, of the phosphorylated intermediates of carbohydrate metabolism, of malate, and of amino acids have been evaluated from measurements of the corresponding subcellular metabolite contents determined using the non-aqueous fractionation technique. Malate, glucose and fructose were found to accumulate in the vacuole, whereas the concentration of sucrose and amino acids in the cytosol were much higher than in the vacuole. The amino acid concentration in the chloroplast stroma is similar to that in the cytosol. Phosphorylated intermediates of carbohydrate metabolism are confined to the chloroplast stroma and the cytosol, confirming the validity of the fractionation method. Whereas triose phosphates and fructose-l,6-bisphosphate are concentrated in the stroma, the concentrations of hexose monophosphates were highest in the cytosol. Since the subcellular metabolite distribution in potato leaves reported here is very similar to that previously described for spinach and barley leaves, we conclude that it may be characteristic for mesophyll cells in general.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Acclimation of potato plants to polyethylene glycol-induced water deficit II. Contents and subcellular distribution of organic solutes

Potato plants (Solanum tuberosum cv. Désirée) were grown hydroponically and subjected to water deficit induced by addition of 10% (w/v) PEG 6000. The potato plants were able to grow under water deficit by accumulating organic solutes (osmoregulation). Osmoregulation occurred in two phases. During the initial 2d hexoses were accumulated, and after 7 d of PEG treatment osmotic adjustment was mostly due to the accumulation of amino acids, especially proline, which accumulated up to 150 times the control content. Sucrose contents remained unchanged in leaves of PEG-treated plants compared with controls, whereas the starch content decreased during PEG treatment.In control leaves, the hexoses and malate were compartmented in the vacuole and sucrose was found in the cytosol and vacuole. Amino acids were distributed between the cytosol and stroma, but only minor amounts of amino acids could be detected in the vacuole. Under water deficit the subcellular distribution of hexoses, malate and sucrose remained unchanged. Most amino acids showed a slight to moderate higher concentration in the vacuole under water deficit. Proline, the metabolite contributing mainly to osmoregulation, was concentrated mostly in the chloroplast and the cytosol. This underlines the important role of proline as the osmolyte under water deficit.     

基于GC-MS的野生和‘鲁赫’刺蔷薇叶片代谢差异研究

为探究野生刺蔷薇（Rosa acicularis）和‘鲁赫’刺蔷薇（Rosa acicularis ‘Luhe’）叶片的代谢物和代谢通路差异，对2种刺蔷薇叶片进行了GC-MS非靶向代谢组学试验，共检测出6种氨基酸、12种糖类、4种烷烃类化合物、17种有机酸、5种酯类和7种其他类化合物，共计51种代谢物。PCA模型符合预期，OPLS-DA模型筛选出19种显著差异代谢物，差异代谢通路分析共分析出5条主要的差异代谢通路。综合各项结果，2种刺蔷薇叶片在部分有机酸和糖类的代谢上有显著差异，同时也在ABC转运蛋白、苯丙烷的生物合成、苯甲酸的降解和丙氨酸、天冬氨酸和谷氨酸的代谢上有显著差异，推测2种植物可能在叶片光合效率和抗胁迫能力上有显著不同，但仍需进一步结合基因组、蛋白质组、转录组和其他方法进行深入研究。     

Cytokinins in the leaves of Ginkgo biloba. II. Metabolism and transport of 8 (14C) zeatin applied to shoot explants

Irrespective of their age, leaves of Ginkgo biloba metabolised applied 8 (¹⁴C) zeatin to compounds of similar chromatographic properties. Glucosylation is apparently not a normal feature of cytokinin metabolism in immature leaves. However, the application of zeatin to these leaves did result in the formation of metabolites which co-chromatographed with glucosylated cytokinins. As far as cytokinin metabolism is concerned therefore, this application of excess zeatin allowed immature leaves to behave as mature or senescing leaves. Overall metabolism was fastest in immature leaves. From the metabolites formed it would appear as if oxidation, which resulted in the formation of a metabolite which co-eluted with N-(purin-6-yl)glycine, was also important in immature leaves. In senescing leaves glucosylation was the major form of metabolism. Extraction and re-application of the polar metabolites (formed from zeatin) to mature leaves resulted in the formation of compounds which co-chromatographed with zeatin. This suggests that these compounds could serve as precursors for zeatin or could be hydrolysed to form zeatin.Very little of the applied radioactivity was exported from the leaves irrespective of their physiological age. When the metabolites, obtained after zeatin application to mature leaves, were extracted and reapplied to the leaves, export of radioactive material was much improved. The results suggest that should cytokinins such as zeatin be translocated to mature leaves of this deciduous gymnosperm their export from the leaves would be unlikely unless first metabolised. In all probability the metabolites concerned are cytokinin glucosides.The financial support of the C.S.I.R., Pretoria, is gratefully acknowledged.     

刺五加和短梗五加叶片初生代谢的发育特异性调控研究

利用GC-MS技术对刺五加（Acanthopanax senticosus（Rupr. et Maxim.） Harms）和短梗五加（Acanthopanax sessiliflorus（Rupr. et Maxim.） Seem）叶片初级代谢产物进行了代谢组学分析,运用PCA和OPLS-DA方法分析后,将刺五加和短梗五加叶片划分为3个发育时期：生长期（Growth period）、旺盛期（Exuberant period）、凋落期（Autumn period）。刺五加叶片3个时期筛选出糖类、氨基酸、有机酸、脂肪酸、多元醇等共53个差异化合物,短梗五加叶片3个时期筛选出糖类、氨基酸、有机酸、脂肪酸、多元醇等共51个差异化合物。进一步分析表明,刺五加和短梗五加叶片在旺盛期和凋落期之间差异化合物最多,其中刺五加主要是糖类、有机酸;短梗五加则除了上述两类化合物外,还包括脂肪酸、多胺类化合物。两者作为同属植物,在差异化合物的组成类型方面具有较高的相似性。结果初步揭示了2种同属药用植物叶片不同时期初级代谢物的积累模式,为2种植物叶片利用提供理论基础。     

Effect of the expression of cyanamide hydratase on metabolites in cyanamide-treated soybean plants kept in the light or dark

Metabolite profiling of untransformed and cyanamide hydratase- (Cah) transformed (denoted 1C) soybean (Glycine max [L.] Merrill) leaves revealed only small differences in plants grown in the greenhouse or in the dark for 24 h, indicating that the Cah enzyme that converts cyanamide to urea has no substrates in soybean leaves and does not affect metabolism. Untransformed leaves sprayed with 0.5% cyanamide developed necrotic lesions within 2 h in the light but not in the dark. The sprayed 1C leaves showed little visible damage and accumulated high concentrations of urea, amino acids, and some sugars, but sucrose decreased over a 24 h period. The untransformed necrotic leaves also accumulated some urea and amino acids apparently due to cyanamide degradation, while sucrose and some organic acids decreased. Sprayed 1C leaves in the dark for 24 h contained very little urea and lower sugar levels. The untransformed sprayed leaves accumulated some organic acids, some sugars including sucrose, and urea and total amino acids. Unsprayed plants of both lines placed in the dark for 24 h showed increases in some amino acids and phosphate, and decreases in other amino acids, sugars, and organic acids. Thus the Cah enzyme can detoxify cyanamide by conversion to urea that is converted to amino acids. Other metabolic changes associated with leaf necrosis and darkness are also described. Principal component analysis confirmed the similarities and differences observed. Comparison of the GC-MS metabolic profiling analysis of amino acids with a dedicated system shows large differences, indicating a limitation of the former system.     

The distribution of organic and mineral components in leaves and stems of Ranunculus fluitans Lam.

The differences in chemical composition of leaves and stems of Ranunculus fluitans Lam. were investigated. Typical distribution of organic and inorganic compounds were generally found not to be influenced by factors as day-time, eutrophication, or age of the weed bed. Starch, sugars, amino acids, and organic acids were at a higher level in the stem, but inorganic cations and anions were accumulated in the leaf. The distribution pattern and the relative contents of individual sugars, acids, and inorganic ions are discussed in detail.     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions

In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.     

Phloem-specific expression of pyrophosphatase inhibits long distance transport of carbohydrates and amino acids in tobacco plants

As reported in a previous paper [Lerchl et al. (1995) Plant Cell, 7, 259–270], expression of Escherichia coli inorganic pyrophosphatase in the cytosol under the control of the phloem-specific rolC promoter from, Agobacterium rhizogenes results in decreased growth of transgenic tobacco plants. In this paper we investigate the effect of the phloem-specific expression of pyrophosphatase on phloem metabolism, and on plant growth and allocation. A small decrease in the hexose phosphate/UDP-glucose ratio, the ATP/ADP ratio and the respiration rate in the midribs of the transformants provides evidence Hint mobilization of sucrose via pyrophosphate-dependent reactions is necessary for phloem energy metabolism. The source leaves of the transformants had higher levels of carbohydrates and amino acids and a much higher glutamine/glutamate ratio than the wild type, showing that export was inhibited and that the growth inhibition was not due to a lack of photoas-similates or organic nitrogen in the leaves. The accumulation of photoassimilates was paralleled by a decrease in photosynthesis, chlorophyll content and ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity, a small increase in hexose phosphates and triose phosphates and a decrease in glycerate 3-phosphate in the source leaves. There was a decrease of soluble sugars and amino acids in sink leaves of the transformants. In sink leaves amino acids decreased more than carbohydrates and a decrease in the glutamine/ glutamate ratio was observed. This was accompanied by a large decrease of nitrate. Sugars and amino acids were also reduced in the root tips of the transformants. The carbohydrate /amino acid ratio decreased 5-fold in the root tips, indicating a particularly smile shortage of carbohydrates. Relatively high levels of sugars and amino acids in the basal regions of the root and the increase in sugars in the midrib indicate that there is also increased leakage of assimilates out of the phloem during long-distance transport. Metabolism is required to maintain phloem function along the transport route, as well as for the initial step of loading. The transformants showed decreased stem and root growth. The growth inhibition was largest in conditions allowing rapid growth of the wild type (high light and nitrogen supply).     

The role of alanine synthesis and nitrate-induced nitric oxide production during hypoxia stress in Cucurbita pepo nectaries

Floral nectar is a sugary solution produced by nectaries to attract and reward pollinators. Nectar metabolites, such as sugars, are synthesized within the nectary during secretion from both pre-stored and direct phloem-derived precursors. In addition to sugars, nectars contain nitrogenous compounds such as amino acids; however, little is known about the role(s) of nitrogen (N) compounds in nectary function. In this study, we investigated N metabolism in Cucurbita pepo (squash) floral nectaries in order to understand how various N-containing compounds are produced and determine the role of N metabolism in nectar secretion. The expression and activity of key enzymes involved in primary N assimilation, including nitrate reductase (NR) and alanine aminotransferase (AlaAT), were induced during secretion in C. pepo nectaries. Alanine (Ala) accumulated to about 35% of total amino acids in nectaries and nectar during peak secretion; however, alteration of vascular nitrate supply had no impact on Ala accumulation during secretion, suggesting that nectar(y) amino acids are produced by precursors other than nitrate. In addition, nitric oxide (NO) is produced from nitrate and nitrite, at least partially by NR, in nectaries and nectar. Hypoxia-related processes are induced in nectaries during secretion, including lactic acid and ethanolic fermentation. Finally, treatments that alter nitrate supply affect levels of hypoxic metabolites, nectar volume and nectar sugar composition. The induction of N metabolism in C. pepo nectaries thus plays an important role in the synthesis and secretion of nectar sugar.     

Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.     

Vacuoles of Mesophyll Cells as a Transient Reservoir for Assimilates

We studied the efflux of radioactive photosynthetic products from the central vacuole into the cytosol of protoplasts isolated from the mesophyll tissue of the sugar beet (Beta vulgaris L.) after their darkening and subsequent cessation of photosynthesis. Among the products accumulated in the vacuole were the ¹⁴C-labelled sugars malate and alanine, small amounts of citric, glutamic, and aspartic acids, and some other amino acids. During the initial 20–30 min of darkness, there was no substantial utilization of photoassimilates accumulated in the vacuole during the preceding light period. An efflux of assimilates occurred later, after 30–40 min of darkness. A decrease in the vacuolar ¹⁴C-sucrose occurred not only due to its exit into the cytosol but also because of its conversion into ¹⁴C-monosaccharides by the vacuolar invertase. In fact, this decrease in the sucrose content correlated well with the accumulation of monosaccharides. Immediately after photosynthesis ceased, the chloroplastic ¹⁴C-starch was utilized for the maintenance of cytoplasmic metabolism. After 30-min darkness, the content of starch in the chloroplasts decreased by several times. We believe that the vacuoles of sugar-beet mesophyll cells are transient reservoirs for assimilates and the products of their conversion (glucose and fructose), which can rapidly leave the vacuole to maintain homeostasis in the cytosol under varying environmental conditions.     

Subcellular volumes and metabolite concentrations in barley leaves

Metabolite concentrations in subcellular compartments from mature barley (Hordeum vulgare L. cv. Apex) leaves after 9 h of illumination and 5 h of darkness were determined by nonaqueous fractionation and by the stereological evaluation of cellular and subcellular volumes from light and electron micrographs. Twenty one-day-old primary leaves of barley with a total leaf volume of 902 μL per mg chlorophyll were found to be composed of 27% epidermis, 42% mesophyll cells, 6% veins, 4.5% apoplast and 23% gas space. While in epidermal cells 99% of the volume was occupied by the vacuole, mesophyll cells with an average volume of 31.3 pL consisted of 23 pL (73%) vacuole, 4.6 pL (19%) chloroplasts, 2.06 pL (6,7%) cytosol (including smaller organelles and vesicles), 0.34 pL (1%) mitochondria and 107 fL (0.34%) nucleus. The differences between leaves harvested after 9 h of illumination and after 5 h of darkness were in the size of the stromal compartment and the starch grains therein. Subcellular metabolite concentrations were calculated from the compartmental volumes and metabolite contents of the compartments as determined by nonaqueous fractionation. The amino-acid concentrations in stroma and cytosol were rather similar after 9 h of illumination and 5 h of darkness. In contrast, the vacuolar amino-acid concentrations were about one order of magnitude lower than the stroma and cytosol values, and there was a slight increase in concentration after 5 h of darkness.     

The effect of nitrate assimilation deficiency on the carbon and nitrogen status of Arabidopsis thaliana plants

Carbon (C) and nitrogen (N) metabolism are integrated processes that modulate many aspects of plant growth, development, and defense. Although plants with deficient N metabolism have been largely used for the elucidation of the complex network that coordinates the C and N status in leaves, studies at the whole-plant level are still lacking. Here, the content of amino acids, organic acids, total soluble sugars, starch, and phenylpropanoids in the leaves, roots, and floral buds of a nitrate reductase (NR) double-deficient mutant of Arabidopsis thaliana (nia1 nia2) were compared to those of wild-type plants. Foliar C and N primary metabolism was affected by NR deficiency, as evidenced by decreased levels of most amino acids and organic acids and total soluble sugars and starch in the nia1 nia2 leaves. However, no difference was detected in the content of the analyzed metabolites in the nia1 nia2 roots and floral buds in comparison to wild type. Similarly, phenylpropanoid metabolism was affected in the nia1 nia2 leaves; however, the high content of flavonol glycosides in the floral buds was not altered in the NR-deficient plants. Altogether, these results suggest that, even under conditions of deficient nitrate assimilation, A. thaliana plants are capable of remobilizing their metabolites from source leaves and maintaining the C–N status in roots and developing flowers.     

Abscisic acid inhibits germination of mature Arabidopsis seeds by limiting the availability of energy and nutrients

The addition of abscisic acid (ABA) to mature non-dormant seeds inhibits their germination. This effect of ABA might be related to its natural function as an endogenous inhibitor of precocious germination during seed formation. In this work, we studied how ABA affects the germination of mature seeds and the growth of nascent seedlings of Arabidopsisthaliana (L.) Heynh. Our findings were as follows: (i) inhibition by ABA was gradual, dose-dependent, and did not disappear after germination; (ii) inhibition of germination was relieved by the addition of metabolizable sugars or amino acids to the plating media; (iii) the effect of sugars and amino acids was cooperative, indicating that these two groups of metabolites relieve different deficiencies; (iv) ABA caused appreciable alterations in energy and nitrogen metabolism; and (v) ABA prevented the degradation of the seed storage proteins. In summary, ABA appears to inhibit seed germination by restricting the availability of energy and metabolites. This mechanism seems consistent with other known effects of ABA. Received: 3 February 1997 / Accepted: 10 March 1997     

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in <Emphasis Type="Italic">Plantago major,Plantago maritima</Emphasis>, <Emphasis Type="Italic">Prunus persica</Emphasis>, and <Emphasis Type="Italic">Apium graveolens</Emphasis>

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.     

Non-morphogenic metabolism during shoot induction in radiata pine cotyledon explants

Summary The effects of excision, light and cytokinin (N⁶-benzyladenine) on¹⁴C-acetate metabolism in cotyledons ofPinus radiata (D. Don) were determined.¹⁴CO₂ was released and the distribution of radioactivity into lipids, sugars, organic acids and amino acids was determined. While light and cytokinin generally caused some increase in metabolism, the effect of excision, i.e., wounding, was most pronounced. Specific metabolites examined (citrate, malate, succinate, alanine, aspartate, glutamate and glutamine) were at least 50% greater in¹⁴C-labeling in excised cotyledons as compared to intact seedlings. This enhancement of wound metabolism would mask possible morphogenically-related changes occurring at that time. This research was supported by the Natural Sciences and Engineering Research Council of Canada Grant A-6467 to T.A. Thorpe.