Termination of nutrient import and development of vein loading capacity in albino tobacco leaves

The sink-source conversion in developing leaves of tobacco (Nicotiana tabacum L.) was studied to determine whether import termination is caused by the onset of export or is related to achievement of positive carbon balance. Albino shoots were grown in vitro and grafted to detopped stems of green tobacco plants. Termination of import was studied by providing mature leaves of the stock plant with ¹⁴CO₂ and detecting the presence of labeled nutrient in developing albino leaves by whole-leaf autoradiography. In albino leaves, import terminated progressively in the basipetal direction at the same stage of development as in leaves of green shoots. Starch was not present in the plastids of mesophyll cells of mature albino leaves but starch was synthesized when discs were cut from these leaves and incubated on 3 millimolar sucrose. Import ceased progressively in developing green leaves even when photosynthesis was prevented by darkening. It was concluded that cessation of import does not require achievement of positive carbon balance and is not the direct result of export initiation.

To determine whether vein loading capacity develops in albino leaves, discs were cut from mature leaves and floated on [¹⁴C]sucrose solution. Uptake of label into the veins was detected by autoradiography and this uptake was sensitive to the phloem loading inhibitor p-chloromercuribenzenesulfonic acid. However, the amount of label taken up by veins in albino leaves was less than that taken up by veins of mature green leaves.

     

Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of ¹⁴C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to ¹⁴CO₂. The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.     

Effect of nitrate infusion into the shoot apoplast on photosynthesis and assimilate transport in symplastic and apoplastic plants

The shoots of fireweed (Chamerion angustifolium (L.) Holub) and common flax (Linum usitatissimum L.) were infused with 50 mM KNO₃ solution to compare the influence of nitrate on photosynthesis and assimilate export from leaves in plants with the symplastic and apoplastic phloem loading, respectively. The infusion of nitrate in the shoots of both plant species lowered ¹⁴CO₂ fixation and enhanced the assimilate transport in the upward direction. Irrespective of the phloem loading type, the incorporation of ¹⁴C into sucrose decreased in nitrate-treated seedlings exposed to assimilation for short (3 min) periods. However, when shoots were sampled 3 h after ¹⁴CO₂ fixation, the content of ¹⁴C-labeled sucrose was higher in treated plants than in control seedlings infused with water. In fireweed, in contrast to flax, a similar temporal pattern was also characteristic for ¹⁴C incorporation into oligosaccharides. Within 3 h after nitrate infusion into the fireweed apoplast, the mitochondria and the cell vacuolar system underwent ultrastructural changes indicative of the increase in cytosolic osmotic pressure. At the same time, we observed accumulation of fibrillar inclusions in cell vacuoles of vascular bundles. It is concluded that the mechanisms of nitrate influence on photosynthesis and sugar export in leaves of symplastic and apoplastic plants are similar to a certain extent and involve the blocking of pores in phloem tubes, initiated by the NO-signaling system.     

Phloem loading in cucumber: combined symplastic and apoplastic strategies

Phloem loading, as the first step of transporting photoassimilates from mesophyll cells to sieve element‐companion cell complex, creates a driving force for long‐distance nutrient transport. Three loading strategies have been proposed: passive symplastic loading, apoplastic loading and symplastic transfer followed by polymer‐trapping of stachyose and raffinose. Although individual species are generally referred to as using a single phloem loading mechanism, it has been suggested that some plants may use more than one, i.e. ‘mixed loading’. Here, by using a combination of electron microscopy, reverse genetics and ¹⁴C labeling, loading strategies were studied in cucumber, a polymer‐trapping loading species. The results indicate that intermediary cells (ICs), which mediate polymer‐trapping, and ordinary companion cells, which mediate apoplastic loading, were mainly found in the fifth and third order veins, respectively. Accordingly, a cucumber galactinol synthase gene (CsGolS1) and a sucrose transporter gene (CsSUT2) were expressed mainly in the fifth/third and the third order veins, respectively. Immunolocalization analysis indicated that CsGolS1 was localized in companion cells (CCs) while CsSUT2 was in CCs and sieve elements (SEs). Suppressing CsGolS1 significantly decreased the stachyose level and increased sucrose content, while suppressing CsSUT2 decreased the sucrose level and increased the stachyose content in leaves. After ¹⁴CO₂ labeling, [¹⁴C]sucrose export increased and [¹⁴C]stachyose export reduced from petioles in CsGolS1i plants, but [¹⁴C]sucrose export decreased and [¹⁴C]stachyose export increased into petioles in CsSUT2i plants. Similar results were also observed after pre‐treating the CsGolS1i leaves with PCMBS (transporter inhibitor). These results demonstrate that cucumber phloem loading depends on both polymer‐trapping and apoplastic loading strategies.     

In vitro and in vivo modification of sugar transport and translocation in celery by phytohormones

In an attempt to address the controversy in the literature as to whether phytohormones have any direct effect on phloem loading of sucrose, we investigated the effect of gibberellic acid (GA₃) and indoleacetic acid (IAA) on sugar transport and translocation in celery (Apium graveolens L. cv. Utah 5270). Both hormones enhanced sucrose uptake into isolated vascular bundles and phloem tissue of celery and enhanced the export of ¹⁴C assimilates from leaves of intact plants in vivo. The hormone-induced increase of uptake into isolated vascular bundles or phloem was specific for sucrose and mannitol which are translocated in phloem. Furthermore, the hormone-induced increase in translocation was not due to an increase in sink demand, since neither glucose nor sucrose uptake rates were affected in the storage parenchyma tissue discs (sink region) in the presence of GA₃ or IAA. The evidence suggests that phytohormones may have a direct effect on phloem loading of sucrose. The possibility of short-term GA₃ and IAA effects on processes resulting in membrane transport of sugars in celery is discussed.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Effect of Exogenously Supplied Foliar Potassium on Phloem Loading in Beta vulgaris L

The effect of foliar application of K⁺ on processes associated with phloem loading was investigated in source leaves of sugar beet (Beta vulgaris L.). KCI was supplied exogenously at concentrations of up to 100 millimolar in the solution bathing the abraded upper epidermis of source leaves. K⁺ added at concentrations below 30 millimolar generally promoted the rate of export of material derived from ¹⁴CO₂ but not from exogenously applied [¹⁴C]sucrose. Paralleling promotion of export, the level of material derived from photosynthesis, which was released into the bathing solution, also increased in response to addition of K⁺ to the free space. Net photosynthetic rate was not affected. K⁺ at 5 and 15 millimolar concentrations did not stimulate uptake of [¹⁴C]sucrose into source leaf discs.     

Companion cell-specific inhibition of the potato sucrose transporter SUT1

In many plants, translocation of sucrose from mesnsophyll to phloem for long-distance transport is carrier-mediated. The sucrose H⁺-symporter gene SUT1 from potato is expressed at high levels in the phloem of mature, exporting leaves and at lower levels in other organs. Inhibition of SUT1 by expression of an antisense gene in companion cells under control of the rolC promoter leads to accumulation of high amounts of soluble and insoluble carbohydrates in leaves and inhibition of photosynthesis. The distribution of in situ localized starch does not correspond with areas of reduced photosynthesis as shown by fluorescence imaging. Dissection of antisense effects on sink and source organs by reciprocal grafts shows that inhibition of transporter gene expression in leaves is sufficient to produce chlorosis in leaves and reduced tuber yield. In contrast to the arrest of plasmodesmal development found in plants that express yeast invertase in the apoplast, in mature leaves of sucrose transporter antisense plants plasmodesmata are branched and have median cavities. These data strongly support an apoplastic mode of phloem loading in potato, in which the sucrose transporter located at the plasma membrane of the sieve element/companion cell complex represents the primary route for sugar uptake into the long-distance translocation pathway.     

A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of ¹⁴CO₂-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate ¹⁴C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE/CC-complex sieve element/companion cell complex We gratefully acknowledge the expert help of Dr. Maarten Terlou, Department of Image Processing and Design, University of Utrecht, in carrying out the densitometric measurements.     

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Inhibition of Loading of C Assimilates by p-Chloromercuribenzenesulfonic Acid : Localization of the Apoplastic Pathway in Vicia faba

The apoplast of mature leaves excised from broadbean (Vicia faba L.) plants was infiltrated with 2 millimolar p-chloromercuribenzenesulfonic acid (PCMBS) via the transpiration stream, and the ability of the tissues to take up sugars was tested. An infiltration time of 75 minutes was sufficient to obtain a maximal (75%) inhibition of exogenous [¹⁴C]sucrose (1 millimolar) uptake. This infiltration affected neither CO₂ assimilation nor the transmembrane potential difference of leaf cells but strongly inhibited phloem loading of endogenous [¹⁴C] assimilates. The study of the symplastic relations between the different cell types of the mature leaf showed that the density of the plasmodesmata is generally very low in comparison with other species investigated so far, particularly when considering the mesophyll/bundle sheath and the bundle sheath/phloem cells connections, as well as the connections of the transfer cell-sieve tube complex with the surrounding cells. These three successive barriers therefore strongly limit the possibilities of symplastic transit of the assimilates to the conducting cells. The comparison of the densities of plasmodesmata in an importing and an exporting leaf suggests that the maturation of the leaf is characterized by a marked symplastic isolation of the phloem, and, within the phloem itself, by the isolation of the conducting complex. As a consequence, these physiological and cytological data demonstrate the apoplastic nature of loading in the mature leaf of Vicia faba, this species undoubtedly presenting a typical model for apoplastic loading.     

Manipulation of sucrose phloem and embryo loading affects pea leaf metabolism,carbon and nitrogen partitioning to sinks as well as seed storage pools

Seed development largely depends on the long‐distance transport of sucrose from photosynthetically active source leaves to seed sinks. This source‐to‐sink carbon allocation occurs in the phloem and requires the loading of sucrose into the leaf phloem and, at the sink end, its import into the growing embryo. Both tasks are achieved through the function of SUT sucrose transporters. In this study, we used vegetable peas (Pisum sativum L.), harvested for human consumption as immature seeds, as our model crop and simultaneously overexpressed the endogenous SUT1 transporter in the leaf phloem and in cotyledon epidermal cells where import into the embryo occurs. Using this ‘Push‐and‐Pull’ approach, the transgenic SUT1 plants displayed increased sucrose phloem loading and carbon movement from source to sink causing higher sucrose levels in developing pea seeds. The enhanced sucrose partitioning further led to improved photosynthesis rates, increased leaf nitrogen assimilation, and enhanced source‐to‐sink transport of amino acids. Embryo loading with amino acids was also increased in SUT1‐overexpressors resulting in higher protein levels in immature seeds. Further, transgenic plants grown until desiccation produced more seed protein and starch, as well as higher seed yields than the wild‐type plants. Together, the results demonstrate that the SUT1‐overexpressing plants with enhanced sucrose allocation to sinks adjust leaf carbon and nitrogen metabolism, and amino acid partitioning in order to accommodate the increased assimilate demand of growing seeds. We further provide evidence that the combined Push‐and‐Pull approach for enhancing carbon transport is a successful strategy for improving seed yields and nutritional quality in legumes.     

Mechanism of export of organic material from the developing fruits of pea

Pisum sativum L. fruits export a small quantity of radiolabeled substances to other plant parts after the fruits are allowed to photosynthesize in the presence of ¹⁴CO₂. Export was uninhibited by peduncle girdling suggesting an apoplastic route for transport of material, presumably by `reverse' flow in the peduncle xylem. To determine if any diurnal water potential gradient formed between pea leaves and fruit might be responsible for the observed export, the water potentials of the various organs were monitored over 24 hours. Water potential differences of up to 7.5 bars existed between leaves and fruit in long photoperiods, and up to 2.5 bars in short photoperiods. Pulses of <sup>14</sup>CO<sub>2</sub> labeling indicated that initial delivery of exported label was to `transpirational sinks,' with subsequent redistribution of label to metabolic sinks. Export to the apical bud appeared to be direct via the xylem. Application of membrane-impermeable inulin to a surgically opened seed coat `cup' resulted in export mainly to the subtending leaf with little redistribution. Simultaneous application of sucrose to the seed coat resulted in more extensive distribution of the sucrose, consistent with reloading of the sucrose into mature leaf phloem. Thus, export of material from fruits appears to occur via a xylem pathway in response to transpirationally derived water potential gradients.     

Enhancement of [C]Sucrose Export from Source Leaves of Vicia faba by Gibberellic Acid

The effect of gibberellic acid (GA₃) on sucrose export from source leaves was studied in broad bean (Vicia faba L.) plants trimmed of all but one source and one sink leaf. GA₃ (10 micromolar) applied to the source leaf, enhanced export of [¹⁴C]sucrose (generated by ¹⁴CO₂ fixation) to the root and to the sink leaf. Enhanced export was observed with GA treatments as short as 35 minutes. When GA₃ was applied 24 hours prior to the ¹⁴CO₂ pulse, the enhancement of sucrose transport toward the root was abolished but transport toward the upper sink leaf was unchanged. The enhanced sucrose export was not due to increased photosynthetic rate or to changes in the starch/sucrose ratio within the source leaf; rather, GA₃ increased the proportion of sucrose exported. After a 10-min exposure to [¹⁴C]GA₃, radioactivity was found only in the source leaf. Following a 2 hour exposure to [¹⁴C]GA₃, radioactivity was distributed along the entire stem and was present in both the roots and sink leaf. Extraction and partitioning of GA metabolites by thin layer chromatography indicated that there was a decline in [¹⁴C]GA₃ in the lower stem and root, but not in the upper stem. This pattern of metabolism is consistent with the disappearance of the GA₃ effect in the lower stem with time after treatment. We conclude that in the short term, GA₃ enhances assimilate export from source leaves by increasing phloem loading. In the long term (24 hours), the effect of GA₃ is outside the source leaf. GA₃ accumulates in the apical region resulting in enhanced growth and thus greater sink strength. Conversely, GA₃ is rapidly metabolized in the lower stem thus attenuating any GA effect.     

Similar photosynthetic response to elevated carbon dioxide concentration in species with different phloem loading strategies

Species have different strategies for loading sugars into the phloem, which vary in the route that sugars take to enter the phloem and the energetics of sugar accumulation. Species with passive phloem loading are hypothesized to have less flexibility in response to changes in some environmental conditions because sucrose export from mesophyll cells is dependent on fixed anatomical plasmodesmatal connections. Passive phloem loaders also have high mesophyll sugar content, and may be less likely to exhibit sugar-mediated down-regulation of photosynthetic capacity at elevated CO₂ concentrations. To date, the effect of phloem loading strategy on the response of plant carbon metabolism to rising atmospheric CO₂ concentrations is unclear, despite the widespread impacts of rising CO₂ on plants. Over three field seasons, five species with apoplastic loading, passive loading, or polymer-trapping were grown at ambient and elevated CO₂ concentration in free air concentration enrichment plots. Light-saturated rate of photosynthesis, photosynthetic capacity, leaf carbohydrate content, and anatomy were measured and compared among the species. All five species showed significant stimulation in midday photosynthetic CO₂ uptake by elevated CO₂ even though the two passive loading species showed significant down-regulation of maximum Rubisco carboxylation capacity at elevated CO₂. There was a trend toward greater starch accumulation at elevated CO₂ in all species, and was most pronounced in passive loaders. From this study, we cannot conclude that phloem loading strategy is a key determinant of plant response to elevated CO₂, but compelling differences in response counter to our hypothesis were observed. A phylogenetically controlled experiment with more species may be needed to fully test the hypothesis.     

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of ¹⁴CO₂ was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either ¹⁴CO₂ or [¹⁴C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [¹⁴C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to ¹⁴CO₂ for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ¹⁴CO₂, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244–1249) indicate that the phloem loading pathway in Coleus is probably symplastic.Abbreviations 3-OMG 3-O-methyl glucose - PCMBS p-chloromercuribenzenesulfonic acid - SE-CCC sieve-element-companion-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose—phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose—phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)^N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.     

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by ¹⁴CO₂ assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total ¹⁴CO₂ fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = −0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.