How the roots contribute to the ability of Phaseolus vulgaris L. to cope with chilling-induced water stress

Intact plants and stem-girdled plants of Phaseolus vulgaris grown hydroponically were exposed to 5 degrees C for up to 4 d; stem girdling was used to inhibit the phloem transport from the leaves to the roots. After initial water stress, stomatal closure and an amelioration of root water transport properties allowed the plants to rehydrate and regain turgor. Chilling augmented the concentration of abscisic acid (ABA) content in leaves, roots and xylem sap. In intact plants stomatal closure and leaf ABA accumulation were preceded by a slight alkalinization of xylem sap, but they occurred earlier than any increase in xylem ABA concentration could be detected. Stem girdling did not affect the influence of chilling on plant water relations and leaf ABA content, but it reduced slightly the alkalinization of xylem sap and, principally, prevented the massive ABA accumulation in root tissues and the associated transport in the xylem that was observed in non-girdled plants. When the plants were defoliated just prior to chilling or after 10 h at 5 degrees C, root and xylem sap ABA concentration remained unchanged throughout the whole stress period. When the plants were chilled under conditions preventing the occurrence of leaf water deficit (i.e. at 100% relative humidity), there were no significant variations in endogenous ABA levels. The increase in root hydraulic conductance in chilled plants was a response neither to root ABA accretion, nor to some leaf-borne chemical signal transported downwards in the phloem, nor to low temperature per se, as indicated by the results of the experiments with defoliated or girdled plants and with plants chilled at 100% relative humidity. It was concluded that the root system contributed substantially to the bean's ability to cope with chilling-induced water stress, but not in an ABA-dependent manner.     

Patterns of Assimilate Production and Translocation in Muskmelon (Cucumis melo L.) : I. Diurnal Patterns

Continuous monitoring of steady-state carbon dioxide exchange rates in mature muskmelon (Cucumis melo L.) leaves showed diurnal patterns of photosynthesis and respiration that were translated into distinct patterns of accumulation and phloem export of soluble sugars and amino acids. Leaf soluble sugar patterns in general followed the pattern of photosynthetic activity observed in the leaf, whereas starch accumulated steadily throughout the light period. Sugar and starch levels declined through the dark phase. Phloem exudate analysis revealed that diurnal levels of the major transport sugars (stachyose and sucrose) in the phloem did not appear to correlate directly with the photosynthetic activity of the leaf but instead were inversely correlated with leaf starch accumulation and degradation. The amino acid pool in leaf tissues remained constant throughout the diurnal period; however, the relative contribution of individual amino acids to the total pool varied with the diurnal photosynthetic and respiratory activity of the leaf. In contrast, the phloem sap amino acid pool size was substantially larger in the light than in the dark, a result primarily due to enhanced export of glutamine, glutamate, and citrulline during the light period. The results indicate that the sugar and amino acid composition of cucurbit phloem sap is not constant but varies throughout the diurnal cycle in response to the metabolic activities of the source leaf.     

Comparison of the contents of sucrose and amino acids in the leaves, phloem sap and taproots of high and low sugar-producing hybrids of sugar beet (Beta vulgaris L.)

The contents of sucrose and amino acids in the leaves, phloemsap and taproots have been analysed in three experimental hybridsof sugar beet and compared with earlier analysed leaf and phloemsap contents in spinach and barley. The three hybrids accumulatedsucrose and amino acids to various extents in the mature rootsas well as in the young taproots (9–12 weeks). The differencesin the sucrose-to-amino acid ratios in the taproots were reflectedin the corresponding ratios in the phloem sap. The leaf contentsof sucrose and amino acids in the three hybrids were found tobe very similar to each other and also to those in spinach andbarley. In contrast, the phloem concentration of sucrose (1.3M) was much higher, and that of amino acids much lower thanin spinach and barley. In the taproots, the overall concentrationof sucrose was about half that in the phloem sap. From thesefindings it is con cluded that the decisive factor in the highsucrose accumulation in sugar beet roots is the very efficientprocess of phloem loading in the leaves. The patterns of theamino acids in the phloem sap and in the taproots resembledthose in the leaves, indicating that there is no special transportform for a-amino nitrogen from the leaves to the roots, butall amino acids which are present in the cytosol are translocated. Key words: Amino acids, Beta vulgaris L., phloem sap, sucrose, tap roots, transport     

Chemical Composition of Phloem Sap from the Uppermost Internode of the Rice Plant

The chemical composition of phloem sap from the uppermost internodeof rice plants (Oryza sativa L., var. Kantou), one week afteranthesis, was compared with that of phloem sap from the leafsheath of a young seedling. The pure phloem sap from rice plantswas collected by an insect laser technique. The phloem sap from the uppermost internode contained a highlevel of sucrose (573.8 mM) which was the only sugar detected.The concentrations of total amino acids, potassium and ATP were124.8 mM, 40.4 mM and 1.76 mM, respectively. The concentrationof sucrose was three times higher and the potassium level wasone third as high in the internode sap as in the phloem sapfrom the leaf sheath. The total concentration of amino acidswas almost the same, but the relative amount of each amino acidwas quite different. The ratios of levels of Glu to Gln andof levels of Asp to Asn in the phloem sap from the uppermostinternode were smaller than those in the phloem sap from theleaf sheath. The adenylate energy charge was 0.92–0.93in both types of phloem sap. The amino acid composition of the phloem sap from the uppermostinternode was compared with that of the phloem sap of the flagleaf and the endosperm sap of the same plant, one week afteranthesis. The differences in composition along the phloem pathwaysuggest the selective translocation of amino acid. (Received July 21, 1989; Accepted December 11, 1989)     

The effect of drought hardening and chilling on ABA content in xylem sap and ABA - delivery rate from root of tomato plant

This paper is a continuation of our studies related to the response of two tomato cultivars: Robin and New Yorker to chilling: the later is more tolerant to chilling than the former one (Starck et al. 1994). The concentration of ABA in the xylem sap and ABA delivery rate (calculated as the amount of ABA exuded in 2h from the cut stump, following shoot removal) were estimated by ELISA. The relative water content (RWC) of the leaf blades and stomatal resistance (RS) were also measured. Tomato plants were grown in a greenhouse, under noncontrolled conditions. Before chilling some of the plants were drought hardened for 10 days (H). As an consequence of water deficit only New Yorker growth slightly decreased. Plants were chilled to 2–5 °C during three consecutive, 16-h nights, preceded by warm days, which caused a decrease in the RWC of leaf blades. Chilling did not decreased leaf blade hydration significantly, but drastically increased the concentration of ABA in the xylem sap in more chilling tolerant cv. New Yorker only. The delivery rate of ABA was markedly enhanced in both cultivars, but much more in New Yorker. Drought hardening increased ABA delivery rate in cv. Robin only, especially after chilling. The lack of correlation between changes in the RWC of leaf blades after low temperature treatment and the concentration of ABA in the xylem sap as well as its delivery rate suggest, that in both tomato cultivars chilling increased ABA level directly, not as an secondery effect of temperature-induced water deficit.     

Apoplastic expression of yeast-derived invertase in potato : effects on photosynthesis, leaf solute composition, water relations, and tuber composition

In potato plants (Solanum tuberosum), a chimeric yeast-derived invertase gene fused to a 35S cauliflower mosaic virus promoter has been expressed. The protein was targeted to the cell wall by using the signal peptide of proteinase inhibitor II fused to the amino terminus of the yeast invertase. The transformed plants had crinkled leaves, showed a reduced growth rate, and produced fewer tubers. Although in the apoplast of the leaves of the transformed plants the content of glucose and fructose rose by a factor of 20, and that of sucrose declined 20-fold, 98% of the carbohydrate in the phloem sap consisted of sucrose, demonstrating the strong specificity of phloem loading. In the leaf cells of the transformed plants, glucose, fructose, and amino acids, especially proline, were accumulated. Consequently, the osmolality of the cell sap rose from 250 to 350 mosmol/kg. Our results show that the observed 75% decrease of photosynthesis is not caused by a feedback regulation of sucrose synthesis and is accompanied by an increase in the osmotic pressure in the leaf cells. In the transformed plants, not only the amino acid to sucrose ratio in the phloem sap, but also the amino acid and protein contents in the tubers were found to be elevated. In the tubers of the transformed plants, the protein to starch ratio increased.     

Diurnal changes in assimilate concentrations and fluxes in the phloem of castor bean (Ricinus communis L.) and tansy (Tanacetum vulgare L.)

Reports about diurnal changes of assimilates in phloem sap are controversial. We determined the diurnal changes of sucrose and amino acid concentrations and fluxes in exudates from cut aphid stylets on tansy leaves (Tanacetum vulgare), and sucrose, amino acid and K(+) concentrations and fluxes in bleeding sap of castor bean pedicel (Ricinus communis). Approximately half of the tansy sieve tubes exhibited a diurnal cycle of sucrose concentrations and fluxes in phloem sap. Data from many tansy plants indicated an increased sucrose flux in the phloem during daytime in case of low N-nutrition, not at high N-nutrition. The sucrose concentration in phloem sap of young Ricinus plants changed marginally between day and night, whereas the sucrose flux increased 1.5-fold during daytime (but not in old Ricinus plants). The amino acid concentrations and fluxes in tansy sieve tubes exhibited a similar diurnal cycle as the sucrose concentrations and fluxes, including their dependence on N-nutrition. The amino acid fluxes, but not the concentrations, in phloem sap of Ricinus were higher at daytime. The sucrose/amino acid ratio showed no diurnal cycle neither in tansy nor in Ricinus. The K(+)-concentrations in phloem sap of Ricinus, but not the K(+) fluxes, decreased slightly during daytime and the sucrose/K(+)-ratio increased. In conclusion, a diurnal cycle was observed in sucrose, amino acid and K(+) fluxes, but not necessarily in concentrations of these assimilates. Because of the large variations between different sieve tubes and different plants, the nutrient delivery to sink tissues is not homeostatic over time.     

Stomatal Closure in Flooded Tomato Plants Involves Abscisic Acid and a Chemically Unidentified Anti-Transpirant in Xylem Sap

We address the question of how soil flooding closes stomata of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants within a few hours in the absence of leaf water deficits. Three hypotheses to explain this were tested, namely that (a) flooding increases abscisic acid (ABA) export in xylem sap from roots, (b) flooding increases ABA synthesis and export from older to younger leaves, and (c) flooding promotes accumulation of ABA within foliage because of reduced export. Hypothesis a was rejected because delivery of ABA from flooded roots in xylem sap decreased. Hypothesis b was rejected because older leaves neither supplied younger leaves with ABA nor influenced their stomata. Limited support was obtained for hypothesis c. Heat girdling of petioles inhibited phloem export and mimicked flooding by decreasing export of [14C]sucrose, increasing bulk ABA, and closing stomata without leaf water deficits. However, in flooded plants bulk leaf ABA did not increase until after stomata began to close. Later, ABA declined, even though stomata remained closed. Commelina communis L. epidermal strip bioassays showed that xylem sap from roots of flooded tomato plants contained an unknown factor that promoted stomatal closure, but it was not ABA. This may be a root-sourced positive message that closes stomata in flooded tomato plants.     

Cucumber mosaic virus infection affects sugar transport in melon plants

Viral infection often affects carbon assimilation and metabolism in host plants. To better understand the effect of cucumber mosaic virus (CMV) infection on sugar transport, carbohydrate levels and the amounts of the various sugars in the phloem sap were determined in infected melon (Cucumis melo L.) plants. Source leaves infected with CMV were characterized by high concentrations of reducing sugars and relatively low starch levels. The altered level of carbohydrates was accompanied by increased respiration and decreased net photosynthetic rates in the infected leaves. Although stachyose was the predominant sugar in phloem sap collected from petioles of control leaves, sucrose (Suc) was a major sugar in the phloem sap of infected leaves. Moreover, analyses of the newly fixed (14)CO(2) revealed a high proportion of radioactive Suc in the phloem sap of infected leaves 60 min post-labeling. The alteration in phloem sap sugar composition was found in source, but not old, leaves. Moreover, elevations in Suc concentration were also evident in source leaves that did not exhibit symptoms or contain detectable amounts of virus particles. The mode by which CMV infection may cause alterations in sugar transport is discussed in terms of the mechanism by which sugars are loaded into the phloem of cucurbit plants.     

Translocation from leaves to fruits of a legume,studied by a phloem bleeding technique: Diurnal changes and effects of continuous darkness

Summary Diurnal changes in the carbohydrates of leaf laminae and fruits and in the bleeding of sugar and amino acids from fruit phloem were followed by successive sampling from a population of Lupinus albus L. plants. Phloem sap was collected for a standard 5 min period from cut distal tips of attached fruits. Daily fluctuations in leaf dry matter resulted largely from changes in starch and sugar. Leaf sugar rose to a maximum in the afternoon, starch to a maximum at, or shortly after, dusk. Leaves lost sugar and starch from dusk to dawn. Phloem bleeding rate varied little over a daily cycle but sucrose levels fluctuated from a noon maximum of 12–13% (w/v) to a dawn minimum of 9–10%. The rhythm of phloem sugar levels matched closely those of fruit and leaf. Phloem amino acid levels fluctuated in phase with that of sucrose: the relative composition of the amino fraction did not vary significantly over the daily cycle. Pulse feeding of source leaves with ¹⁴CO₂ at different times in the photoperiod allowed study of the pattern of release of labelled photosynthate to the fruit phloem and the build up and depletion of ¹⁴C starch in leaves. Plants transferred to continuous darkness showed a rapid decline in output and concentration of phloem sap solutes, and translocated nitrogen to their fruits at only one quarter of the rate of control plants retained in natural daylight. The combined data from the experiments showed that the rate of output of sugar from cut phloem of a fruit was directly related to the current level of sugar in leaves. When leaf sugar levels were low (5–10 mg ml tissue water^-1) sugar in phloem was 10–11 times more concentrated than in source leaves, but at high leaf sugar levels (25–30 mg ml^-1) this concentration difference was only 3–4 fold.     

Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit,leaves and phloem exudate

Abscisic acid (ABA) was identified by combined gas liquid chromatography-mass spectrometry in sieve-tube exudate collected from the cut stylar ends of white lupin fruit. Water stress caused an increase in ABA levels in leaf, seed and pod tissues and phloem exudate. When compared with levels in extracts of these tissues, the concentration of ABA in sieve-tube sap was very high. It is suggested that ABA is actively transported out of mature leaves in the phloem and this finding is discussed in terms of the ABA balance of the plant.Abbreviations ABA abscisic acid - GLC gas liquid chromatography     

Sugar accumulation in roots of two grape varieties with contrasting response to water stress

Root sugar accumulation was studied in two grapevine varieties contrasting in tolerance to water stress. During a 10‐day water withholding treatment, the drought‐tolerant variety, Grenache, sustained less negative predawn and midday leaf water potentials as well as root water potential compared with the sensitive variety, Semillon. Grenache vines also maintained lower stomatal conductance and transpiration than Semillon vines throughout the drying period. In both varieties there was accumulation of sucrose in the roots and concentrations were inversely correlated to leaf and root water status. In both Grenache and Semillon, elevated root osmolality was associated with decreased soil moisture indicating that sugar accumulation may play a role in osmotic protection. Petiole xylem sap abscisic acid (ABA) concentrations increased with water deficit in both varieties and were highest for vines with the most negative root and predawn leaf water potentials. Furthermore, root sucrose concentrations were positively correlated with leaf xylem sap ABA concentrations, indicative of integration between carbohydrate metabolism and the ABA signalling system. Similar root sugar accumulation patterns between the two varieties, however, demonstrate that other factors are likely influencing the ability of the drought‐tolerant variety to remain hydrated.     

Phloem loading in peach: Symplastic or apoplastic?

Sorbitol and sucrose are the two main soluble carbohydrates in mature peach leaves. Both are translocated in the phloem, in peach as in other rosaceous trees. The respective role of these two soluble carbohydrates in the leaf carbon budget, and their phloem loading pathway, remain poorly documented. Though many studies have been carried out on the compartmentation and export of sucrose in sucrose-transporting species, far less is known about sorbitol in species transporting both sucrose and sorbitol. Sorbitol and sucrose concentrations were measured in several tissues and in sap, in 2-month-old peach (Prunus persica L. Batsch) seedlings, i.e. leaf blade, leaf main vein, petiole, xylem sap collected using a pressure bomb, and phloem sap collected by aphid stylets. The sorbitol to sucrose molar ratio depended on the tissue or sap, the highest value (about 7) found in the leaf main vein. Sorbitol concentration in the phloem sap was about 560 mM, whereas that of sucrose was about 140 mM. The lowest sorbitol and sucrose concentrations were observed in xylem sap collected from the shoot. The volume of the leaf apoplast, estimated by infiltration with ³H-inulin, represented about 17% of the leaf blade water content. This volume was used to calculate a global intracellular concentration for each carbohydrate in the leaf blade. Following these simplifying assumptions, the calculated concentration gradient between the leaf's intracellular compartment and phloem sap is nil for sorbitol and could thus allow for the symplastic loading of the phloem of this alditol. However, infiltration of ¹⁴C-labelled source leaves with 2 mMp-chloromercuribenzenesulfonic acid (PC-MBS), a potent inhibitor of the sucrose carrier responsible for phloem loading in sucrose-transporting plants, had a significant effect on the exudation of both labelled sucrose and sorbitol from the phloem. Therefore, in peach, which is a putative symplastic loader according to minor vein anatomy and sorbitol concentration gradients, apoplastic loading may predominate.     

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.     

An <i>in Vitro</i> Approach to Investigate the Role of Abscisic Acid in Alleviating the Negative Effects of Chilling Stress on Banana Shoots

Banana is a tropical crop cultivated in warm places. Chilling stress in Egypt is making banana crops less productive. Abscisic acid (ABA), a key plant hormone, regulates metabolic and physiological processes and protects plants from a variety of stresses. In vitro growing banana shoots were pre-treated with ABA at four concentrations (0, 25, 50, and 100 mM) and chilled at 5°C for 24 h, followed by a six-day recovery period at 25°C. By comparing ABA treatments to both positive and negative controls, physiological and biochemical changes were investigated. Chilling stress (5°C) caused a considerable increase in lipid peroxidation and ion leakage and reduced photosynthetic pigments in cold-treated plantlets. Increasing the concentration of ABA to 100 µM enhanced the response to chilling stress. ABA had a major effect on mitigating chilling injury in banana shoots by keeping cell membranes stable and lowering the amount of ion leakage and lipid peroxidation. Also, ABA significantly maintained the photosynthetic pigment concentration of banana shoots; accumulated higher amounts of total soluble carbohydrates and proline; and increased DPPH radical scavenging activity. Furthermore, ABA treatment enhanced cold tolerance in chilling-stressed banana shoots through the regulation of antioxidant enzyme activity. Overall, the results show that ABA is a good choice for protecting banana shoots from the damage caused by chilling stress.     

Abscisic acid in apoplastic sap can account for the restriction in leaf conductance of white lupins during moderate soil drying and after rewatering

We studied the effects of drought on leaf conductance (g) and on the concentration of abscisic acid (ABA) in the apoplastic sap of Lupinus albus L. leaves. Withholding watering for 5d resulted in complete stomatal closure and in severe leaf water deficit. Leaf water potential fully recovered immediately after rewatering, but the aftereffect of drought on stomata persisted for 2d. ABA and sucrose were quantified in pressurized leaf xylem extrudates. We assumed that the xylem sucrose concentration is negligible and hence that the presence of sucrose in leaf extrudates indicated that they were contaminated by phloem. To eliminate this interference, the concentration of ABA in leaf apoplast was estimated by extrapolation to zero sucrose concentration, using the regression between ABA and sucrose concentrations. The estimated apoplastic ABA concentration increased by 100-fold with soil drying and did not return to pre-stress values immediately following rewatering. g was closely related to the concentration of ABA in leaf apoplast. Furthermore, the feeding of exogenous ABA to leaves detached from well-watered plants brought about the same degree of depression in g as resulted from the drought-induced increase in ABA concentration. We therefore conclude that the observed changes in the concentration of ABA in leaf apoplast were quantitatively adequate to explain drought-induced stomatal closure and the delay in stomatal reopening following rewatering.     

Influence of phloem transport, N-fertilization and ion accumulation on sucrose storage in the taproots of fodder beet and sugar beet

To investigate the factors governing the accumulation of sucroseand amino acids in the taproots of sugar beet, their contentswere measured in the leaves, phloem sap and the taproots ofsugar beet, fodder beet and a hybrid between both, grown oneither 3.0 or 0.5 mM nitrate. In the taproots the contents ofmalate, citrate and inorganic ions were also determined. Forthe high sucrose accumulation in sugar beet as compared to theother varieties three factors were found. (a) In sugar beet,less amino acids and more sucrose are taken up into the phloemthan in fodder beet. (b) In sugar beet, the sucrose and aminoacid syntheses are less sensitive to the nitrate concentrationsthat are required for optimal plant growth than in other varieties.In fodder beet, upon raising the nitrate concentration from0.5 mM to 3 mM, the synthesis and storage of sucrose is decreasedand that of amino acids increased. The corresponding valuesin sugar beet (0.5 mM) are similar to those in fodder beet andare not much affected by an increase of nitrate. (c) The sucroseaccumulation is limited by the accumulation of inorganic ionsin the taproots. The sucrose content in the taproots is negativelycorrelated to the total ion content. Whereas sucrose representstwo-third of all solutes in the taproots of sugar beet, it amountsto only one-third of the solutes in fodder beet taproots. Key words: Amino acids, Beta vulgans L, phloem sap, potassium, sucrose storage, sugar beet, taproots, transport     

Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth

Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [¹⁴C]stachyose or [¹⁴C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [¹⁴C]stachyose, but some degree of vein labeling with [¹⁴C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and α-galactosidase and extensively metabolized exogenously supplied ¹⁴C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species.