Auxin-induced assimilate translocation in the bean stem (Phaseolus vulgaris L.)

Decapitation of the fully-elongated fourth internode of Phaseolus vulgaris plants resulted in the disappearance from the internode of soluble acid invertase (EC 3.2.1.26). This loss was prevented by local applications to the internode of indol-3yl-acetic acid (IAA) and, at the point of IAA application, the specific activity of the enzyme increased by up to 3 times its initial value within 48 h of treatment. IAA applications stimulated the acropetal translocation to the internode of ¹⁴C-sucrose applied to the subtending (second) trifoliate leaf 30 h after decapitation and the start of the auxin treatment. Labelled assimilates accumulated in the IAA-treated region of the internode. Following decapitation the concentration of hexose sugars in the internode fell and that of sucrose rose substantially, but these trends were reversed by IAA treatment. However, small local accumulations of sucrose occurred at the point of auxin application where tissue concentrations of IAA were greatest (determined using [1-¹⁴C] IAA).Considerable quantities of starch were present in the ground parenchyma of the internodes at the start of the experiment but, in the absence of IAA, this was remobilised within 48 h of decapitation. IAA prevented starch loss at and below its point of application to the internode, but not from more distal tissues. Cambial proliferation, radial growth and lignification were stimulated in and below IAA-treated regions of the internode. These observations are discussed in relation to the hormonal regulation of assimilate translocation in the phloem.     

Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

Plasticity in sunflower leaf and cell growth under high salinity

A group of sunflower lines that exhibit a range of leaf Na ⁺ concentrations under high salinity was used to explore whether the responses to the osmotic and ionic components of salinity can be distinguished in leaf expansion kinetics analysis. It was expected that at the initial stages of the salt treatment, leaf expansion kinetics changes would be dominated by responses to the osmotic component of salinity, and that later on, ion inclusion would impose further kinetics changes. It was also expected that differential leaf Na ⁺ accumulation would be reflected in specific changes in cell division and expansion rates. Plants of four sunflower lines were gradually treated with a relatively high (130 mm NaCl) salt treatment. Leaf expansion kinetics curves were compared in leaves that were formed before, during and after the initiation of the salt treatment. Leaf areas were smaller in salt‐treated plants, but the analysis of growth curves did not reveal differences that could be attributed to differential Na⁺ accumulation, since similar changes in leaf expansion kinetics were observed in lines with different magnitudes of salt accumulation. Nevertheless, in a high leaf Na⁺‐including line, cell divisions were affected earlier, resulting in leaves with proportionally fewer cells than in a Na⁺‐excluding line. A distinct change in leaf epidermal pavement shape caused by salinity is reported for the first time. Mature pavement cells in leaves of control plants exhibited typical lobed, jigsaw‐puzzle shape, whereas in treated plants, they tended to retain closer‐to‐circular shapes and a lower number of lobes.     

Loading of assimilates in wheat leaves. II. The path from chloroplast to vein

Abstract. An improved method is described for micro-autoradiography of water-soluble substances alter freeze-substitution of plant tissue in which water is rigorously excluded. Resin sections are cut and flattened dry, and dry photographic emulsion is mounted on them. When the location of ¹⁴C in wheat leaves after assimilation of ¹⁴CO₂ was studied with this method, it was found that ¹⁴C entered the intermediate veins before the laterals and entered both types of veins along the flanks of the veins adjacent to the phloem. High concentrations of ¹⁴C were found in small spaces in the cells of mesophyll and vein parenchyma; these spaces coincide with the nuclei. The concentration of ¹⁴C in these nucleus-associated spaces was as high as that reached at later times in the sieve tubes. Water washed the ¹⁴C out of these spaces of the sections and the label in the washings was predominantly in sucrose. The high ¹⁴C concentrations of the nucleus-associated spaces were particularly easily leached. It is concluded that the raising of the sugar concentration to the high levels found in sieve tubes can take place in these leaves in a special space in each mesophyll cell not, or not only, at the boundary of the sieve tubes.     

Electron-Microscopic Evidence of the Vacuolar Nature of Phloem Exudate

We performed electron-microscopic examination of structural diurnal changes in the lumen of sieve tubes and the vacuolar system of corresponding companion cells and changes induced by the experimental blockage of assimilate export from the leaf by its cold-girdling. For these investigations, Cucurbita pepo L. and Helianthus annuus L. plants were used, that is, plant species from groups of symplastic and apoplastic plants, which differ in the type of companion cells and a mode of phloem terminal loading. The examinations showed the complete identity of changes in the electron texture of the sieve-tube lumens and companion-cell vacuoles in both plant species in the course of a day, when the level of assimilates changed, or after export blockage. Similar changes in the structure of the vacuolar labyrinths were stated in the companion cells under normal conditions and after cold-girdling, as related to the rate of sieve-tube loading with the vacuolar exudate. Vacuolar expansion and starch accumulation developing in response to changes in the assimilate level in the evening and after cold blockage of the assimilate export occurred in different types of cells, as dependent on their position in the symplast domains. However, the rate of the process similarly depended on the balance between assimilate synthesis and export. Synchronous changes in the texture of the sieve-tube lumen and companion-cell vacuoles were observed within each complex, but asynchronous changes occurred in different complexes. We suggested this phenomenon for recognizing the particular complexes, when they are grouped in a bundle. We observed no signs of cytoplasm or protein synthetic machinery in the sieve tubes. We concluded that the sieve-tube lumen and vacuoles of companion cells are common in nature. Similar electron texture of the images of the companion-cell vacuolar labyrinth and tube lumens, their connection through the lateral sieve fields, morphological modifications of the companion-cell vacuolar system as dependent on the activity of sieve tube loading—all of these facts imply the continuity of these transport compartments and fluxes in them and the similarity in the composition of the exudates from companion-cell vacuoles and phloem tubes.     

Sequential response of whole plant water relations to prolonged soil drying and the involvement of xylem sap ABA in the regulation of stomatal behaviour of sunflower plants

Sunflower plants ( Helianihus animus cv. Tall Single Yellow} were grown in the greenhouse in drain pipes (100 mm inside diameter and 1 m long) rilled with John Innes No. 2 compost. When the fifth leaf had emerged, half of the plants were left unwatered for 6 days, rewatered for 2 days and then not watered for another 12 days. Measurements of water relations and abaxial stomatal conductance were made at each leaf position at regular intervals during the experimental period. Estimates were also made of soil water potentials along the soil profile and of ABA concentrations in xylem sap and leaves.
Soil drying led to some reduction in stomatal conductance alter only 3 days but leaf turgors were not reduced until day 13 (6 days after rewatering). When the water relations of leaves did change, older leases became substantially dehydrated while high turgors were recorded in younger leaves. Leaf ABA content measured on the third youngest leaf hardly changed over the first 13 days of the experiment, despite substantial soil drying, while xylem ABA concentrations changed very significantly and dynamically as soil water status varied, even when there was no effect of soil drying on leaf water relations. We argue that the highest ABA concentrations in the xylem, found as a result of substantial soil drying, arise from synthesis in both the roots and the older leaves, and act to delay the development of water deficit in younger leases.
In other experiments ABA solutions were watered on to the root systems of sunflower plants to increase ABA concentrations in xylem sap. The stomatal response to applied ABA was quantitatively very similar to that to ABA generated as a result of soil drying. There was a log-linear relationship between the reduction of leaf conductance and the increase of ABA concentration m xylem sap.     

Transfer cells and solute uptake in minor veins of Pisum sativum leaves

Morphometric and physiological studies were conducted to determine whether the wall ingrowths of transfer cells in the minor-vein phloem of Pisum sativum L. leaves increase the capacity of the cells for solute influx. Size and number of wall ingrowths are positively correlated to the photon flux density (PFD) at which the plants are grown. An analysis of plasmodesmatal frequencies indicated that numerous plasmodesmata are present at all interfaces except those between the sieveelement-transfer-cell complex (SE-TCC) and surrounding cells where plasmodesmata are present but few in number. Flux of exogenous sucrose into the SE-TCC was estimated from kinetic profiles of net sucrose influx into leaf discs, quantitative autoradiography, and measurements of sucrose translocation. Flux based both on the saturable (carrier-mediated) and the linear components of influx was 47% greater in leaves of plants grown at high PFD (1000 mol·m^–2·s^–1) than those grown in low PFD (200 mol·m^–2·s^–1) and was paralleled by a 47% increase in SE-TCC plasmalemma surface area. Flux of endogenous photosynthate across the SE-TCC plasmalemma was calculated from carbon balance and morphometric data. The increase in flux in high-light leaves over that in low-light leaves can be explained on the basis of an increase in plasmalemma surface area. In intact leaves, a standing osmotic gradient may facilitate transport of solute into transfer cells with extensive wall elaborations.Abbreviations LPI leaf plastochron index - PCMBS p-chloromercuribenzenesulfonic acid - PFD(s) photon flux density (densities) - SE-TCC sieve-element-transfer-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competitive Grant 90000854, and Hatch funds.     

Effects of nitrate on influx, efflux and translocation of potassium in young sunflower plants

Influx, efflux and translocation of K⁺(⁸⁶Rb) were studied in the roots of sunflower seedlings ( Helianthus annuus L. cv. Uniflorus) treated with 0–4.0 m M NO₃⁻ during a 9 day growth period or a 24 h pretreatment period. Roots treated with high levels of NO₃⁻ absorbed and translocated more K⁺(⁸⁶Rb) than seedlings treated with low levels of NO₃⁻. The content of K⁺ in the shoots was, however, higher in seedlings treated with low levels of NO₃⁻, indicating a low rate of retranslocation of K⁺ in those plants. K⁺(⁸⁶Rb) efflux was highest into the low-NO₃⁻ solutions. All effects on K⁺(⁸⁶Rb)-fluxes were more obvious in high-K plants than in low-K plants. The results are discussed in relation to the Dijkshoorn-Ben Zioni hypothesis for K⁺+ NO₃⁻-uptake and translocation in plants.     

Purification and characterisation of an inhibitor of a cathepsin B-like proteinase from sunflower seed

Cathepsin B is a vitally important enzyme in various physiological processes and in tumor invasion and metastasis. A cathepsin B inhibitor, HCB-SunI, was identified and purified from sunflower seeds, Helianthus annuus, using ammonium sulfate precipitation and two steps of conventional chromatography. The molecular mass of HCB-SunI was estimated to be 12 kDa by SDS-PAGE and 12.32 kDa by MALDI TOF MS. Its N-terminal amino acid sequence was determined to be: PYGGGGTESG. HCB-SunI not only inhibited Helicoverpa cathepsin B (HCB) but also decreased the growth of HeLa and glioma cells by 7 ～ 27% and 6 ～ 22%, respectively, when the cells were grown in a final concentration of 0.002 ～ 0.008 μM inhibitor.     

Investigation of the limitations to photosynthesis induced by leaf water deficit in field-grown sunflower (Helianthus annuus L.)

Abstract. The diurnal cycling of leaf water potential (Ψ_leaf) in field-grown sunflower ( Helianthus annuus ) was used to investigate the cause of water deficitinduced limitation of net photosynthesis. Daily midafternoon decreases in Ψ_leaf of up to 1.5 MPa and in net photosynthesis of up to 50% were typical for irrigated sunflower during seed filling. These midafternoon values were lowered an additional 0.6 to 0.8 MPa by prolonged drought treatment. There was a nearly linear relationship between the decline in net photosynthesis and reductions in leaf conductance over the course of the day. Thus, it was unexpected to find that the low, midafternoon rates of photosynthesis were associated with the highest intercellular CO₂ concentrations. These and other observations suggest that the daily decline in photosynthesis represents a 'down regulation' of the biochemical demand for CO₂ that is coordinated with the diurnally developing need to conserve water, thus establishing a balanced limitation of photosynthesis involving both stomatal and non-stomatal factors. There were no indications that either short term (i.e. diurnal declines in Ψ_leaf) or long term (i.e. drought treatment) water deficits caused any damage or malfunctioning of photosynthesis. Rather, both the daily declines in photosynthesis and the nearly 25% decrease in leaf area induced by prolonged drought appeared to be well-controlled adaptive responses by field-grown sunflower plants to limited water availability.     

FITC-dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves

The liquid in the free space of leaf cell walls, the apoplast, is in direct contact with the plasma membrane and its nutrient uptake systems. Therefore, the pH of the apoplast is of utmost interest. We have elaborated a non-destructive method by which excised sunflower leaves ( Helianthus annuus cv. Erika) were perfused with fluorescein isothiocyanate-dextran (FITC-dextran) (4 000 Da) via the transpiration stream. We showed that leaf apoplast pH can be measured by using the fluorescence ratio technique together in conjunction with this dye. Evidence is provided that FITC-dextran does not penetrate the plasma membrane over a period of ca 17 h from the beginning of dye perfusion. Dye enrichment in the leaf apoplast did not cause an 'inner filter effect' and thus the fluorescence ratio was only dependent on pH. In vivo calibration yielded a pKa of 5.92, which was virtually identical to the pKa of 5.93 calculated for dye solutions. Hence, FITC-dextran can be detected in complex environments and covers a pH range prevailing in the leaf apoplast.
Based on this method we developed a microscope image technique visualizing pH gradients between various cell types. The pH in the lumen of the xylem vessel was ca 0.3–0.5 units lower than that of the apoplast of surrounding cells. Nitrate present in the leaf apoplast caused an increase in pH, especially in the dark. Under these conditions, in the intercostal area, the apoplast pH around the stomata was ca 0.5–1.0 units higher than that of the surrounding epidermal cells.     

In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots

The effect of boron excess and deficiency on H⁺ efflux from excised roots from sunflower ( Heliarahus annuus L. cv. Enano) seedlings and on plasma membrane H⁺-ATPase (EC 3.6.1.35) in isolated KI-washed microsomes has been investigated. When seedlings were grown in media with toxic levels of H₃BO₃ (5 m M ) or without added boron and exposed to light conditions, an inhibition of the capacity for external acidification by excised roots was observed as compared to roots from seedlings grown with optimal H₃BO₃ concentration (0.25 m M ). Toxic and deficient boron conditions also inhibited the vanadate-sensitive H⁺-ATPase of microsomes isolated from the roots. The mechanism of boron toxicity was investigated in vitro with microsorne vesicles. A strong effect of boron on the vanadate-sensitive, ATP-dependent H⁺ transport was found, but the vanadate-sensitive phospho-bydrolase activity was not affected. These results suggest that boron could exert an effect on the plasma membrane properties, directly or indirectly regulating, proton transport.     

Short-term effects of boron, germanium and high light intensity on membrane permeability in boron deficient leaves of sunflower

With the ultimate purpose of clarifying the mechanism for aluminium (Al) toxicity and for Al tolerance, we tried to isolate cDNAs whose expression is induced by Al treatment and phosphate (P_i) starvation. We performed P_i starvation and Al treatment (two-step treatment) on suspension-cultured cells of Nicotiana tabacum L. cv. Samsun and then constructed a cDNA library using poly(A)⁺-RNA derived from the treated cells. Four independent cDNA clones (pAL 111, 139, 141 and 142) were isolated from the library by differential screening. Northern blot hybridization analysis indicated that the expression of these clones was induced by P_i starvation. Furthermore, we found that pAL 111 and pAL 142 are also induced by Al treatment. The complete cDNA sequencing of these 4 clones was determined. The results indicated that pAL111 is identical to the parA gene of N. tabacum, which is described as an auxin-regulated gene and that pAL142 is highly homologous to the parB gene of N. tabacum whose product has glutathione S-transferase (GST, EC 2.5.1.18) activity. Furthermore, we found a cysteine-rich domain in the amino acid sequence of pAL139. No DNA and deduced amino acid sequences homologous to the pAL141 were found.     

Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.     

Effects of leaf position and nitrogen supply on the expansion of leaves of field grown sunflower (Helianthus annuus L.)

Leaf growth responses to N supply and leaf position were studied using widely-spaced sunflower plants growing under field conditions. Both N supply (range 0.25 to 11.25 g added N per plant) and leaf position significantly (p=0.001) affected maximum leaf area (LA_max) of target leaves through variations in leaf expansion rate (LER); effects on duration of expansion were small. Specific leaf nitrogen (SLN, g N m^-2) fell quite rapidly during the initial leaf expansion phase (LA < 35% LA_max) but leveled off during the final 65% increase of leaf area. This pattern held across leaf positions and N supply levels. Leaf nitrogen accumulation after 35% LA_max continued up to achievement of LA_max; reductions in the higher SLN characteristic of the initial phase were insufficient to cover the nitrogen requirements for expansion during the final phase. LER in the quasi-linear expansion phase (35 to 100% of LA_max) was strongly associated with SLN above a threshold that varied with leaf position (mean 1.79±0.225 g N m^-2). This contrasts with the response of photosynthesis at high irradiance to SLN, which has previously been shown to have a threshold of 0.3 g N m^-2; in the present work saturation of photosynthetic rate was evident when SLN reached 1.97 g N m^-2. Thus, once the area of a leaf exceeds 35% of LA_max, expansion proceeds provided SLN values are close to the levels required for maximum photosynthesis. However, growth of leaves during the initial expansion phase ensures a minimum production of leaf area even at low N supply levels.     

Leaf expansion of four sunflower (Helianthus annuus L) cultivars in relation to water deficits. I. Patterns during plant development

Abstract. The influence of a slow stress and recovery cycle on the pattern of leaf expansion in four diverse sunflower cultivars ( Helianthus annuus L. cvs. Hysun 31, Havasupai, Hopi and Seneca) was studied in a glasshouse. Stress had no significant effect on the time of flower bud emergence and anthesis, or on final leaf number, but delayed the appearance of leaves at high insertions in all cultivars except Hysun 31.
Leaf expansion was markedly reduced as the predawn leaf water potential decreased from −0.35 to −0.60 MPa, and the predawn turgor pressure decreased from 0.3 to 0.2 MPa, and expansion ceased at a predawn leaf water potential of about −1.0 MPa, i.e. when the predawn turgor pressure reached zero.
The leaves most reduced in final size when water was withheld were those at the insertions which grew the most rapidly in unstressed plants. The maximum reduction in final leaf size of 25–35% was similar in all cultivars and was due to retardation of the rate of leaf expansion: the duration of leaf expansion was actually increased by stress. However, leaves that were initiated during stress, but emerged after rewatering, had final leaf areas at least equal to those in the unstressed plants: in the cultivar Seneca, the final size of leaves of high insertion was significantly greater in stressed than unstressed plants, whereas in the three other cultivars the final leaf sizes were similar in both treatments. All four cultivars examined adjusted osmotically to the same degree, but leaf water potentials in one, Seneca, increased more rapidly after rewatering than in the other three, and this may have contributed to the greater relative leaf size in the leaves of high insertion in this cultivar.     

Does light-promoted export from Commelina benghalensis leaves result from differential light-sensitivity of the cells in the mesophyll-to-sieve tube path?

In contrast to the light-promoted uptake by mesophyll cells, light slightly inhibited sucrose uptake by stripped leaf disks of Commelina benghalensis L. This phenomenon appeared to result from a light-promoted vein-associated release, as light stimulated photosynthate release from stripped disks and inhibited that from mesophyll cells. In the -presence of the resorption-blocker p -chloromercuriphenylsulfonic acid, (PCMBS) the release of preloaded [¹⁴C]-sugars (sucrose, glucose) and [¹⁴C]-amino acids (alanine, asparagine, proline, valine, α-aminoisobutyric acid) from stripped disks was doubled in the light. Illumination enhanced by 20 to 60% the release of endogenous leaf cell compounds (sucrose, H₂PO-₄, K⁺, Mg²⁺, Ca²⁺) from stripped disks in the presence of PCMBS. Light also increased the export of [¹⁴C]-assimilates from intact leaves by 20% after pulse-labelling with ¹⁴CO₂. A model for loading is proposed, based on the differential light sensitivities of the plasma membranes in the mesophyll-to-sieve tube path.     

Responses of photosynthesis and carbohydrate-partitioning to limitations in nitrogen and water availability in field-grown sunflower*

Abstract. Sunflower plants (Helianthus annuus L., cv. CGL 208) were field-grown in adjacent plots of varying resource availability. Control plants received irrigation (on a 4–5 d interval) and high levels of fertilizer nitrogen. Nutrient-stress (N-stress) plants received control levels of irrigation but no nutrient amendments and were determined to be nitrogen-limited. Water-stress (H₂O-stress) plants received control levels of fertilizer nitrogen, but no irrigation after approximately 6 weeks of plant growth. Both stress treatments reduced maximum and diurnal net photosynthesis (A) but resulted in different physiological or biochemical adjustments that tended to maintain or increase A per unit of resource (nitrogen or water) in shortest supply while decreasing the ratio of A per unit of abundant resource. Nutrient-stress reduced total foliar nitrogen, foliar chlorophyll, and initial and total RuBPCase activities, thereby enhancing or preserving photosynthetic nitrogen-use efficiency (NUE), defined as the maximum A observed per unit of leaf nitrogen, relative to the control and H₂O-stress treatments. In addition, N-stress reduced photosynthetic water-use efficiency (WUE), defined as the ratio of A to stomatal conductance to water vapour (g). The slope of A versus g increased with H₂O-stress. In addition, sunflower plants responded to H₂O-stress by accumulating foliar glucose and sucrose and by exhibiting diurnal leaf wilting, which presumably provided additional improvements in photosynthetic WUE through osmoregulation and reduction of midday radiation interception respectively. Photosynthetic NUE was decreased by H₂O-stress in that control levels of total nitrogen, foliar chlorophyll, and RuBPCase activities were maintained even after mean diurnal levels of A had fallen to less than 50% of the control level. We conclude that field-grown sunflower manages a trade-off between photosynthetic WUE and NUE, increasing use efficiency of the scarce resource while decreasing use efficiency of the abundant resource.