Increased Expression of a Phloem Membrane Protein Encoded by NHL26 Alters Phloem Export and Sugar Partitioning in Arabidopsis

The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield. Similar effects were observed when NHL26 was overexpressed in companion cells, under the control of a companion cell–specific promoter. The soluble sugar content of the phloem sap and sink organs was lower than that in the wild type, providing evidence of a sugar export defect. This was confirmed in a phloem-export assay with the symplastic tracer carboxyfluorescein diacetate. Leaf sugar accumulation was accompanied by higher organic acid, amino acid, and protein contents, whereas analysis of the metabolite profile of phloem sap exudate revealed no change in amino acid or organic acid content, indicating a specific effect on sugar export. NHL26 was found to be located in the phloem plasmodesmata and the endoplasmic reticulum. These findings reveal that NHL26 accumulation affects either the permeability of plasmodesmata or sugar signaling in companion cells, with a specific effect on sugar export.     

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.     

Response of Phloem Loading and Export to Rapid Changes in Sink Demand

Sink demand was abruptly changed for an illuminated sugar beet source leaf by shading the six to ten other source leaves. Export of recently assimilated, labeled material underwent a transient increase and then returned to a steady rate approximately equal to the pretreatment rate. Uncovering the darkened leaves caused a transient decrease in export of ¹⁴C; following recovery there was a gradual decline. It remains to be established whether export of unlabeled reserves occurs in response to increased sink demand. The possibility that phloem loading increases in response to decreased sieve tube turgor was tested. Phloem loading of exogenous ¹⁴C-sucrose increased when turgor in leaf cells was decreased by floating leaf discs on solutions with up to 1 M mannitol osmoticum. However, the increase appeared to be the result of plasmolysis of mesophyll cells possibly resulting from easier access to minor veins via the free space. Phloem loading in leaf discs continued undiminished even though sieve tube-companion cell sucrose concentration exceeded a calculated value of 1 M. Regulation of export to meet sink demand by a direct response of phloem loading to a turgor or concentration set point does not appear to occur. Phloem loading may be promoted by the influx of water which drives mass flow, increasing phloem loading in response to increased velocity of transport.     

Leaf Export and Partitioning Changes Induced by Short-Term Inhibition of Phloem Transport

Both electric shock and cold shock applied to the phloem pathway,and known to induce temporary inhibition to long-distance phloemtransport, are shown to have an immediate and sustained effectupon both export of carbon from the leaf and its partitioningbetween alternative sinks. The changes in export occurred byan unknown mechanism, while the changes in partitioning areinterpreted as responses to changed export Key words: Carbon partitioning     

Importance of Pollen and Senescent Petals in the Suppression of Alfalfa Blossom Blight (Sclerotinia sclerotiorum) by Coniothyrium minitans

The effect of pollen and senescent petals on the suppression of alfalfa (Medicago sativa L.) blossom blight (Sclerotinia sclerotiorum) by the mycoparasite Coniothyrium minitans was investigated. When incubated at 20°C for 39 h, germination of conidia of C. minitans and ascospores of S. sclerotiorum was 99.9 and 98.6%, respectively, in the presence of alfalfa pollen (9×10⁴ pollen grains mL^?1), whereas spore germination of both organisms was <0.5% in the absence of pollen (in water). In the presence of a commercial pollen product, Swiss? pollen granules (mainly bee pollen), germination was 99.6% for C. minitans and 98.3% for S. sclerotiorum when the pollen concentration was 1.0% (w/v). When the pollen concentration was reduced to 0.1% (w/v), germination was reduced to 13.0% for C. minitans and 10.8% for S. sclerotiorum. Tests on detached alfalfa florets showed that the colonization of alfalfa florets by S. sclerotiorum was significantly suppressed by C. minitans in the presence of pollen (1.0% Swiss? pollen granules), especially when C. minitans was inoculated 1-day before S. sclerotiorum. In vivo inoculation tests revealed that the efficacy of C. minitans in the protection of alfalfa pods from the infection by S. sclerotiorum was affected by the time at which C. minitans was applied. When C. minitans was applied on young blossoms of alfalfa at the anthesis stage, pod infection was 96.6% for the treatment of C. minitans+S. sclerotiorum and 99.6% for the treatment of S. sclerotiorum alone. However, when C. minitans was applied on senescent petals of alfalfa at the pod development stage, pod infection was 8.0% for the treatment of C. minitans+S. sclerotiorum compared to 90.8% for the treatment of S. sclerotiorum alone. These results suggest that timing of the application of C. minitans is critical for the mycoparasite to compete with S. sclerotiorum for the source of nutrients from pollen and senescent petals, and for its control of alfalfa blossom blight caused by S. sclerotiorum.     

An Effect of Ethylene on the Distribution of 14C-sucrose from the Petals to Other Flower Parts in the Senescent Cut Inflorescence of Dianthus caryophyllus

The distribution of carbon-14 in the flower parts of the cutcarnation inflorescence after feeding ¹⁴C-sucrose through thepetals was studied during natural ageing and after ethylenetreatment. Levels of ethylene which caused irreversible wiltingof petals also promoted an accelerated transfer of the radioactivesucrose to the nectar, gynaecium and stem. Since the nectarreceived a relatively large proportion of the radioactive carbon,the composition of the sugars in the nectar and the vascularizationof the nectary were investigated. Sucrose comprised about 85per cent of the nectar sugars and the balance was glucose andfructose. The vascular tissue closest to the nectary consistedof phloem elements; tracheary elements terminated deeper inthe receptacle and were surrounded by a ring of phloem. Thepercentage of solutes in the nectar was about 18 per cent andincreased when the flowering stems were placed in sucrose solutions;the solutes in the nectar were principally sugars. Taken togetherthe results show that the nectary can act as a sink for sucroseand, in the flower at least, that translocation of sucrose takesplace in the phloem. The results provide further evidence forthe hypothesis that ethylene promotes mobilization of substrateand an efflux of material from petals to the gynaecium, nectarand stem.     

Plantlet Production from Morphogenetically Competent Cell Suspensions of Daylily

Cell suspensions of the diploid daylily cultivar Autumn Blazewere produced from larger masses of tissue by culture in thebasal medium of Murashige and Skoog supplemented with 10 percent v/v coconut water and 2 mg 1^–12,4–D. By drasticallylowering the level of 2,4–D, followed by transferral toa modified White's or Schenk and Hildebrandt medium, clustersgrow and ultimately give rise to embryonic structures. A finalperiod in a semi-solid medium stimulates shoot and root growthto the point where successful transplanting of plantlets tosoil is assured provided safeguards to prevent ‘dampingoff’ and desiccation are taken. Normal plantlet formationmay be arrested in the formation of neomorphs which do not seemper se to be capable of further development but they can giverise to morphologically normal plantlets after they are stimulatedto form callus which, in turn, is given an appropriate sequenceof stimuli. Hemerocallis, daylily, totipotent cells, micropropagation, tissue culture.     

The Relationship between Sugar and Amino Acid Export to the Phloem in Young Wheat Plants

The relationship between amino acid and sugar export to thephloem was studied in young wheat plants (Triticum aestivumL. ‘Pro-INTA, Isla Verde’) using the EDTA-phloemcollection technique. Plants grown with a 16 h photoperiod showeda rapid decrease in the concentration of sugars and amino acidsin the phloem exudate from the beginning of the dark period.When plants grown with a 16 h photoperiod were kept in the darkfor longer than 8 h the free amino acid content in leaves andexudate (on a dry weight basis) increased continually throughoutthe 72 h of darkness. During the first 24 h of darkness thesugars in the phloem exudate decreased to 30% of the initialvalue, and returned to the control level when plants were returnedto light. When plants grown under low light intensity for 10d were transferred to high light intensity, they showed an increasein leaf sugar content (dry weight basis) after 3 d but therewere no differences in leaf free amino acid content (dry weightbasis) compared to low-light plants. The sugar concentrationin the phloem exudate was increased by higher light intensities,but there was no difference in the amino acid concentrationof the phloem exudate, and thus the amino acid:sugar ratio inthe phloem decreased in the high-light plants. The present resultssuggest that amino acids can be exported to the phloem independentlyof the export of sugars. Copyright 1999 Annals of Botany Company Sugar exudation, amino acid transport, nitrogen, phloem, transport, wheat, Triticum aestivum L.     

Recovery of Totipotent Cells and Plantlet Production from Daylily Protoplasts

The release of protoplasts by means of enzymes from a totipotentcell suspension of a diploid daylily (Hemerocallis cv. ‘AutumnBlaze’), their collection and distribution in plasticculture vessels and their subsequent regeneration is described.Attention is given to aggregation and to the different formsof growth during the initial stages of culture. The methodsfor the subsequent multiplication of the regenerated cells intomorphogenetically competent compact clusters and the manipulationof these clusters to yield embryo-plantlets are also outlined.The implications of all this in terms of potential for the geneticmodification of daylily is discussed. Hemerocallis, cloning, protoplasts     

Physiological Properties of Abscission Accelerator from Senescent Leaves

A substance which is not abscisic acid accelerates leaf abscission by regulating ethylene production.     

Karotype Analysis of a Daylily Clone Reared from Aseptically Cultured Tissues

Ann Bot 47, 121–131, 1981 In the course of shorteningand consolidating data of Table 3, values for centromeric indexwere deleted The column wrongly labelled centromeric index provides,in fact, the arm ratios. If necessary, centromeric indexes canbe calculated algebraically from the figures provided (cf Levan,Frega and Sandberg, 1965, p 205)     

Karyotype Analysis of a Daylily Clone Reared from Aseptically Cultured Tissues

Hemerocallis cv. ‘Autumn Blaze’ plantlets generatedin quantity on semi-solid media from tiny nodules asepticallymaintained and multiplied in liquid were raised to maturity.Comparisons were made between phenotypic characters of floweringplants derived from culture and a non-cultured clonal populationwhich included the plant from which the primary explant of thematerial was taken. Plants were pheno-typically identical. Withinthe limits of the cytological techniques used, it was not possibleto disclose differences between karyotype of root tip cellsof plants produced via aseptic culture and those that had notbeen cultured. Hemerocallis, daylily, karyotype analysis, cloning, tissue culture     

Senescent phenotypes of skin fibroblasts from patients with Tangier disease

Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) approximately 10 compared with control cells. TD cells practically ceased proliferation at PDL approximately 30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated beta-galactosidase (SA-beta-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients.     

Altered Composition of Cerebral Microvessel Membrane Phosphoglycerides from Senescent Mouse

Abstract— Phosphoglyceride and fatty acid composition was determined in the cellular membranes of isolated cerebral microvessels and brain parenchymal cells (neurons and glia) taken from 10-, 20-, and 27–30-month-old C57BL6/NNIA mice. Lipids were extracted from each fraction and the fatty acid profiles of ethanolamine, cho-line, serine, and inositol phosphoglycerides analyzed by gas chromatography. The results suggest that membrane phosphoglycerides from cerebral microvessels are significantly more affected by the aging process than are those of the brain parenchyma. Relative percentage for fatty acids in cerebral microvessels indicate an overall decline in membrane unsaturation with a concomitant elevation in the level of saturation. The decline in unsaturation is reflected primarily in the loss of precursor fatty acids for arachidonic (18:2n-6 and 20:3n-6) and docosahexaenoic (20:5n-3 and 22:5n-3) acids. Levels of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids in each phos-phoglyceride remained unchanged with age; however, mol% for ethanolamine plasmalogen, a major source of these fatty acids, was significantly reduced in 27–30-month-old mice. Conversely, mol% for choline phospho-glyceride increased with age. The age-related changes in fatty acid profile for microvessel membrane phosphoglycerides are reflected by increased saturation/unsaturation ratios and decreased unsaturation indices. These parameters were not affected by aging in parenchymal membranes.     

Phloem mobility of magnesium

Magnesium-28 was applied to specific leaves of bean (Phaseolus vulgaris) and barley (Hordeum vulgare) plants. After 24 hours, as much as 7% of the absorbed Mg was exported from the treated bean leaves and 11% was transported basipetally from the treated zone of the barley leaves. Transport of Mg did not occur past a heat-killed section of the treated leaf, thereby indicating that translocation took place via the phloem. Mg movement in the phloem was also evident in autoradiograms of bean stem segments in which the xylem was separated from the phloem by a thin sheet of plastic.     

The Abscission of Rose Petals

Petal abscission was studied in twelve hybrid tea rose (Rosahybrida L.) cultivars. At about 20 °C the time to petalabscission in uncut stems in greenhouses was the same as incut stems placed in water in the greenhouse or in a climate-controlledroom. The time between petal unfolding and abscission dependedon the cultivar, and varied between 12 and 35 d. The time topetal abscission of the cultivars was inversely correlated withtheir flower diameter at full bloom (linear regression, r² =0·82). In the cultivars with a relatively large flowerdiameter (10-18 cm) the petals fell without visible desiccationsymptoms, whereas in the group with a small diameter the petalswere partially or fully desiccated when shed. Fertilization occurred in some flowers of a few cultivars studied.In cultivars with a relatively large flower diameter (Papa Meilland,Cocktail, Dr. Verhage, Tineke) it had no effect on the timeto abscission in Motrea, Europa, and Carolien roses, which bearsmall flowers, the petals fell after fertilization, whereasin unfertilized flowers of the latter group of cultivars anabscission zone just above the uppermost node became activeand all parts above this node (pedicel and flower) turned brownand desiccated, though remained attached for more than a month. It is concluded that in the cultivars investigated: (a) thetime to petal abscission was inversely related to their flowerdiameter, (b) abscised petals were more desiccated in cultivarsin which the time to abscission was longer, (c) fertilizationhad little effect on the time to abscission in most cultivars,whereas the absence of fertilization prevented petal abscissionin a number of the small-diameter cultivars where it was replacedby flower abscission, and (d) cutting and placement in waterat 20 °C did not affect the time to abscission.Copyright1995, 1999 Academic Press Abscission, fertilization, flowers, petals, Rosa hybrida L., rose, water stress, carbohydrate stress     

Phloem exudation from castor bean: Induction by massage

Summary Exudation can be induced or greatly enhanced in Ricinus by massaging the stem periodically some time before tapping the sap by making an incision. Nevertheless temporary massage during exudation stops flow completely indicating that flow is prevented normally by a pressure sensitive sealing mechanism which becomes desensitised by regular massage.     

Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume

The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N₂. Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K⁺ content and high ratio of Mg²⁺ to Ca²⁺. Fruit cryopuncture sap became labeled with ¹⁴C following feeding of [¹⁴C]urea to leaves or adjacent walls of the fruit, of ¹⁴CO₂ to the pod gas space, and of [¹⁴C] asparagine or [¹⁴C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of ¹⁴C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of ¹⁴C distribution in phloem sap suggested that [¹⁴C]allantoin was metabolized to a greater extent in its passage to the fruit than was [¹⁴C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO₃-fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO₃-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of ¹⁵NO₃ to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant.     

Enhancement of Phloem exudation from cut petioles by chelating agents

The photosynthetic assimilates in leaves of Perilla crispa attached to the plant were labeled by treating the leaves with (14)CO(2). When subsequently detached, these leaves exuded a negligible amount of radioactivity from the cut petiole into water. Ethylenediaminetetraacetate (EDTA), citric acid, and ethyleneglycol-bis (beta-aminoethyl ether) N,N'-tetraacetate greatly increased exudation of labeled assimilates into a solution bathing the petioles. The optimal concentration of EDTA was 20 mm, and maximal exudation took place between 2 and 4 hours after excision. Up to 22% of the radioactivity fixed in the leaf was exuded into an EDTA solution as compared to an export of 38% from attached leaves. The amount of radioactivity in the exudate was much reduced at low temperature. Presence of EDTA was required in the collecting solution for only 1 to 2 hours; upon transfer to water, exudation continued as in continuous presence of EDTA. Ca(2+) completely inhibited the effect of EDTA.Anatomical studies indicated that callose formation on the sieve plates near the cut surface of the petioles was less in leaves on EDTA than on water.More than 95% of the radioactivity exuded by detached leaves was present in the sugars verbascose, stachyose, raffinose, and sucrose, which are translocated in the phloem of Perilla. Labeled glucose, fructose, and galactinol were detected in the leaf blade and petiole, but not in exudates.The addition of EDTA to a solution bathing the petiole of detached leaves of Chenopodium rubrum and Pharbitis nil also increased the exudation of labeled assimilates. In these two species, label appeared only in a compound that cochromatographed with sucrose.It is concluded that the radioactive products in the solution are actually exuded by the phloem. Possibly EDTA chelates Ca(2+) that otherwise participates in the reactions that seal cut phloem.