Dinitrophenol-induced efflux of sucrose from maize scutellum cells

Maize scutellum slices accumulated sucrose during incubation in glucose, fructose or sucrose. Sucrose was accumulated in two compartments, tentatively     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.     

Sucrose Efflux from the Maize Scutellum

Sucrose efflux from maize scutellum slices was promoted by high pH and by K+, Na+ or Rb+. Incubation in mannose (which drastically reduces the ATP level) caused high rates of sucrose efflux only when KCl was present at pH 8. The effects of triphenylmethylphosphonium ion (TPMP+, a lipid soluble cation) on sucrose efflux were similar to those of mannose plus KCl. Mannose and TPMP+ caused release of stored sucrose into the cytoplasm, but pH8 and KCl (mannose) or pH 8 (TPMP+) in the bathing solution were necessary for rapid efflux of sucrose. Rb⁺ uptake took place during sucrose efflux. In mannose, rates of Rb⁺ uptake and sucrose efflux were low at pH 5.6 and high at pH 8.0, although the time courses for uptake and efflux were different. It is concluded that sucrose efflux is electrogenic and that it occurs as sucrose-H⁺ symport. A scheme for sucrose transport across plasmalemma and tonoplast is presented.     

Sucrose transport and hexose release in the maize scutellum

Since hexoses readily diffuse from maize scutellum cells, it should be possible to detect them if they are produced during sucrose transport at the tonoplast or the plasmalemma. To test this idea, scutellum slices were placed in dinitrophenol (DNP) (which inhibits hexose utilization while greatly increasing utilization of vacuolar sucrose), and the utilization, uptake and leakage of sugars were measured. Only negligible amounts of hexose appeared in the DNP solution during a 5-hr incubation during which the slices metabolized 72μmol of sucrose. Glucose and fructose, added at a concentration of 2 mM, were taken up by the slices at rates 33% and 14% (respectively) of the rate of vacuolar sucrose utilization. It is suggested, therefore, that sucrose transport at the tonoplast does not release free hexose into the cytoplasm. Sucrose transport at the plasmalemma was studied using DNP- and mannose-treated slices. During incubation of these slices in sucrose, the disappearance of sucrose resulted in the appearance of significant quantities of glucose and fructose in the bathing solution. Evidence is presented that sucrose is split into glucose and fructose during transport across the plasmalemma. It is concluded that free hexose is not normally a product of this splitting but is a result of an uncoupling in the transport system caused by the DNP or mannose treatments.     

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.     

14C sucrose efflux from the perimedulla of growing potato tubers

Abstract An experimental system has been developed for studying efflux of ¹⁴C assimilates in growing potato tubers. Small wells are cut into the phloem-rich perimedulla and filled with trap solutions of varying composition which inhibit or promote assimilate efflux. One well on each tuber acts as the treatment while a second well acts as the control. Movement of ¹⁴C into wells occurred at comparable rates to that found in intact tissue, harvested from importing tubers in the form of microcores. Sucrose was the predominant translocated sugar in the stolon and was not hydrolysed in either the wells or the microcores following unloading. Efflux into wells containing agar traps was stimulated 40-fold relative to buffer controls by the addition of 20 mol m^?3 EGTA to the agar. This was interpreted as passive efflux to the apoplast due to increased membrane permeability in the pathway between the sieve elements and the collecting wells. The EGTA stimulation was reversed by addition of Ca²⁺. ¹⁴C efflux into buffered solutions was inhibited significantly by both DNP and PCMBS, suggesting the involvement of active and carrier-mediated transport components. However, it was not possible to determine whether these compounds acted at the site of unloading only, or on the short-distance transfer step between phloem and collecting wells. The rate of tracer efflux was not significantly different when 1 mol m^?3 and 300 mol m^?3 sucrose were applied to the wells, indicating insensitivity of solute movement to low apoplastic solute concentrations. However, raising the solute concentration to 800 mol m^?3 caused a severe inhibition of tracer efflux. These results were duplicated with mannitol as the osmoticum. It is suggested that plasmolysis prevented further efflux by disruption of a predominantly symplastic transport pathway between the phloem and collecting wells.     

Effect of an increased sink demand on the carbon metabolism and export of a maize source leaf

The sink demand was increased on a source maize leaf ( Zea mays L. cv. F7F2) by darkening all the leaves except the fourth, which was maintained under the prevailing irradiance conditions. The parameters of carbon metabolism were measured precisely during the first hours, and then daily during one week. The ambient photosynthetic activity and the maximum photosynthetic capacity were not altered by the treatment but the soluble carbohydrate and starch contents diminished, while ADP-glucose pyrophosphorylase (EC 2.7.7.27) activity increased. The carbon export rate, evaluated by the rate of disappearance of radioactivity after a 1-min ¹⁴CO₂ pulse, was faster than in control leaves. A compartmental analysis of the time course of ¹⁴C export further indicated that the sucrose pool providing the export flux was largely increased by the dark treatment. The darkened leaf 5, taken as an example of the darkened sources, was completely depleted of its carbohydrate content after one day in the dark and remained devoid of carbohydrates during the following week.     

The role of mesophyll cell tonoplast in determining the route of phloem loading. Thirty years of the studies of phloem loading

The evidence of light, electronic, and confocal microscopy collected within the 30-year period is reviewed to revise the concept of assimilate loading in phloem. It is the starting point located in mesophyll cells, which determines the route of assimilate export from mesophyll to phloem, rather than its final segment located in the terminal phloem. Plastids, photosynthesis, and the primary pool of photosynthates are localized in the vacuome of mesophyll cells. All chemicals applied to leaf surface are loaded to phloem via apoplast, even in the symplastic plants. It follows that photoassimilates are not loaded via apoplast because they cannot leave mesophyll and not due to the lack of pumps and transporters in the terminal phloem cells. Of two membranes separating vacuome and apoplast, the tonoplast confers the barrier function. The impossibility to overcome this barrier raises the hydrostatic pressure in the vacuome to the level that induces plasmodesma development between the cells. With the loss of tonoplast barrier function for assimilates, the latter leave for apoplast, this process is incompatible with building the vacuolar loading route. Two alternative mechanisms of phloem loading diverge initially because of different barrier functions of tonoplast. The radical change in these functions makes up the crucial advantage of the young group of apoplastic dicot plants (about 20 000 species), whose evolution is associated with expansion of meadow-steppe vegetation 5–7 million years ago. Such change would evolve due to the climate differentiation in the late myocene period, when heat and moisture were lacking at vast territories. A large group of temperate herbs evolved and expanded because of these changes in the assimilate compartmentalization.     

玉米雄性不育资源的发掘与利用

植物雄性不育是指植物雄性生殖器官不能产生正常有功能花粉的现象.玉米(Zea mays L.)是重要的粮食作物之一,也是较早利用杂种优势的作物之一.当前,生产上广泛种植的玉米品种类型主要是单交种.我国玉米杂交种的播种面积常年稳定在6.2亿亩左右,年用种量10亿公斤以上,常年制种面积高达250多万亩.利用传统的人工去雄或机...     

Alkaline cooking and tortilla quality in maize grains from the humid,tropical lands of Mexico

Maize (Zea mays L.) tortilla is the major staple food for the Mexican population. Nine tropical maize genotypes were evaluated. All samples had white grains, a common characteristic in tropical maize, and therefore they were appropriate for nixtamalized flour industry. Grain, flour, masa and tortilla characteristics of each maize genotype were evaluated. Length, width, thickness, weight of 1000 grains and hardness of grain were determined. Moisture content, proteins, fat, ash, mean particle size, water absorption index, enthalpy, and flour temperature were also evaluated. Adhesiveness and cohesiveness were evaluated in masa. Moisture content, protein, capacity to puff up, roll making, tension and cutting strength were determined in tortillas. There were significant differences (p≤0.05) in most of the evaluated characteristics. Grain length values varied between 9.26 and 11.02 mm for populations 23 and 22, respectively. Grain hardness oscillated between 11.17 (population 32) and 14.75 (landrace Mejen). According to the weight of 1000 grains most genotypes had small grains. The minimum and maximum moisture values of flour and tortillas were 8.33-9.99% and 46.20-50.36%, respectively. The texture of tortillas elaborated from population 32 and landrace Mejen had the lowest tension and cutting strength, resulting the best genotypes for making tortilla.     

玉米及马齿苋叶片SOD活性诱导研究

以玉米幼苗及马齿苋作材料,通过甘露醇、H2O2、臭氧和强光等胁迫后,用NBT光化还原抑制法测定叶中SOD活性的变化。臭氧和强光能诱导玉米叶片SOD活性增加。0.5mol／L甘露醇处理玉米叶片12h,SOD活性上升,至48h后下降;在该甘露醇溶液中另外加入10^-2mol/L H2O2;处理12h后SOD活性基本不变。对玉米叶片单独用10^-2mol/L H2O2诱导,30min内SOD活性上升到最高值,随着处理时间的延长又逐渐下降。用耐强光、耐干旱的野生马齿苋作为材料,与玉米相比,其叶片SOD基础活性低于后者;若予以正午强光结合渗透胁迫2h,其叶中SOD活性增幅超过玉米,从种间比较的角度旁证了SOD在抗逆性中的作用。推测植物中存在比活性氧更为直接的物理诱导机制。     

Transpiration inhibition by stored xylem sap from well-watered maize plants

There is increasing evidence that a chemical signal exists in xylem sap of plants subjected to water deficits which influences physiological responses in plant shoots. An important method of studying this signal is the transpiration response of excised leaves exposed to xylem sap collected from plants. However, Munns et al [Plant, Cell & Environment 16, 867–877] cautioned that transpiration inhibition is observed when xylem sap collected from wheat and barley is stored before determining physiological activity. The objective of the study reported here was to determine if transpiration inhibition develops in maize sap collected from well-watered plants when the sap is stored under various conditions. It was found that storage of maize sap collected from well-watered plants for only 1 d at -20°C resulted in the development of substantial transpiration inhibition in bioassay leaves. Storage of sap at 4°C resulted in the development of the effect after 2 weeks, while storage at ?86°C showed only small transpiration inhibition after 3 weeks. The major source of the transpiration inhibition was the development of a substance in the stored sap that resulted in physical blockage of the transpiration stream in bioassay leaves. However, a small signal component may also have developed in the stored sap. Because of the possibility of ionic activity under freezing conditions at ?20°C, calcium was studied for its potential involvement in the transpiration inhibition. However, the calcium concentrations found to inhibit transpiration were nearly an order of magnitude larger than the calcium concentrations observed in xylem sap.     

Acid phosphatase from maize scutellum: Succinylation and some kinetic properties of the active enzyme

Acid phosphatase purified from maize scutellum did not dissociate into subunits upon acylation with succinic anhydride. The enzyme maintained its catalytic activity after succinylation of 52 free amino groups permolecule. The results also showed that free amino groups may play an important role in the maintenance of enzyme stability at pH values greater than 5.4.     

Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop

Background and Aims

Floral development depends on photosynthetic products delivered by the phloem. Previous work suggested the path to the flower involved either the apoplast or the symplast. The objective of the present work was to determine the path and its mechanism of operation.

Methods

Maize (Zea mays) plants were grown until pollination. For simplicity, florets were harvested before fertilization to ensure that all tissues were of maternal origin. Because sucrose from phloem is hydrolysed to glucose on its way to the floret, the tissues were imaged and analysed for glucose using an enzyme-based assay. Also, carboxyfluorescein diacetate was fed to the stems and similarly imaged and analysed.

Key Results

The images of live sections revealed that phloem contents were released to the pedicel apoplast below the nucellus of the florets. Glucose or carboxyfluorescein were detected and could be washed out. For carboxyfluorescein, the plasma membranes of the phloem parenchyma appeared to control the release. After release, the nucellus absorbed apoplast glucose selectively, rejecting carboxyfluorescein.

Conclusions

Despite the absence of an embryo, the apoplast below the nucellus was a depot for phloem contents, and the strictly symplast path is rejected. Because glucose and carboxyfluorescein were released non-selectively, the path to the floret resembled the one later when an embryo is present. The non-selective release indicates that turgor at phloem termini cannot balance the full osmotic potential of the phloem contents and would create a downward pressure gradient driving bulk flow toward the sink. Such a gradient was previously measured by Fisher and Cash-Clark in wheat. At the same time, selective absorption from the apoplast by the nucellar membranes would support full turgor in this tissue, isolating the embryo sac from the maternal plant. The isolation should continue later when an embryo develops.     

Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L.

Minor-vein ultrastructure and sugar export were studied in mature summer and winter leaves of the three broadleaf-evergreen species Ajuga reptans var. artropurpurescens L., Aucuba japonica Thunb. and Hedera helix L. to assess temperature effects on phloem loading. Leaves of the perennial herb Ajuga exported substantial amounts of assimilates in form of raffinose-family oligosaccharides (RFOs). Its minor-vein companion cells represent typical intermediary cells (ICs), with numerous small vacuoles and abundant plasmodesmal connectivity to the bundle sheath. The woody plants Hedera and Aucuba translocated sucrose as the dominant sugar species, and only traces of RFOs. Their minor-vein phloem possessed a layer of highly vacuolated cells (VCs) intervening between mesophyll and sieve elements. Depending on their location and ontogeny, VCs were classified either as companion or parenchyma cells. Both cell types showed symplasmic continuity to the adjacent mesophyll tissue although at a lower plasmodesmal frequency compared to the Ajuga ICs. p-Chloromercuribenzenesulfonic acid did not reduce leaf sugar export in any of the plants, indicating a symplasmic mode of phloem loading. Winter leaves did not show symptoms of frost injury, and the vacuolar pattern in ICs and VCs was equally prominent in both seasons. Starch accumulation as a result of reduced phloem loading was not observed to be triggered by low temperature. In contrast, high amounts of starch were found in mesophyll and bundle-sheath cells of summer leaves. Physiological data on season-dependent leaf exudation showed the maintenance of sugar export in cold-acclimated winter leaves.     

Comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines A188 and A632

Induction, maintenance, differentiation and embryogenic capacity of callus obtained from immature embryos by culture on induction medium, proliferation medium, maturation medium and regeneration medium, respectively, were compared for two inbred lines of maize, i.e. A188 and A632. The callus of inbred line A188 was embryogenic and maintained embryogenic capacity for at least 1 year. Immature embryos of inbred line A632 formed callus that was not embryogenic. It only produced roots. When sucrose was replaced by sorbitol to induce or improve embryogenesis, again only A188 formed embryogenic callus. Subculture of this callus, however, allowed 4 week intervals in stead of 2 week intervals without loss of embryogenic capacity. When A188 was pollinated with A632 pollen, embryogenic callus was obtained from cultured immature "F₁" embryos, showing that embryogenic capacity was inherited, maternally. The callus did not differ from the embryogenic callus generated on selfed A188 embryos. When A632 was pollinated with A188 pollen, embryogenic callus was obtained too, showing that embryogenic capacity was also inherited paternally, though the embryogenic capacity diminished quickly, and the stability of the callus was lower than in the reciprocal cross. This revised version was published online in June 2006 with corrections to the Cover Date.