The Use of Fluorescent Tracers to Characterize the Post-Phloem Transport Pathway in Maternal Tissues of Developing Wheat Grains

Various polar fluorescent tracers were used to characterize the pathways for apoplastic and symplastic transport in the "crease tissues" (i.e. the vascular strand, chalaza, nucellus, and adjacent pericarp) of developing wheat (Triticum aestivum L.) grains. With mostly minor exceptions, the results strongly support existing views of phloem unloading and post-phloem transport pathways in the crease. Apoplastic movement of Lucifer yellow CH (LYCH) from the endosperm cavity into the crease was virtually blocked in the chalazal cell walls before reaching the vascular tissue. However, LYCH could move slowly along the cell wall pathway from the chalaza into the vascular parenchyma. Slow uptake of LYCH into nucellar cell cytoplasm was observed, but no subsequent symplastic movement occurred. Carboxyfluorescein (CF) imported into attached grains moved symplastically from the phloem across the chalaza and into the nucellus, but was not released from the nucellus. In addition, CF moved in the opposite direction (nucellus to vascular parenchyma) in attached grains. Thus, the post-phloem symplastic pathway can accommodate bidirectional transport even when there is an intense net assimilate flux in one direction. When fresh sections of the crease were placed in fluorochrome solutions (e.g. LYCH or pyrene trisulfonate), dye was rapidly absorbed into intact cells, apparently via unsealed plasmodesmata. Uptake was not visibly reduced by cold or by respiratory inhibitors, but was greatly reduced by plasmolysis. Once absorbed, the dye moved intercellularly via the symplast. Based on this finding, a size-graded series of fluorescein-labeled dextrans was used to estimate the size-exclusion limits (SEL) for the post-phloem symplastic pathway. In most, and perhaps all, cells of the crease tissues except for the pericarp, the molecular diameter for the SEL was about 6.2 nm. The SEL in much of the vascular parenchyma may be smaller, but it is still at least 3.6 nm. Channel diameters would likely be about 1 nm larger, or about 4.5 to 7.0 nm in the vascular parenchyma and 7.0 nm elsewhere. These dimensions are substantially larger than those for "conventional" symplastic connections (about 3 nm), and would have a greater than proportionate effect on the per channel diffusive and hydraulic conductivities of the pathway. Thus, relatively small and probably ultrastructurally undetectable adjustments in plasmodesmatal structure may be sufficient to account for assimilate flux through the crease symplast.     

Ethylene-Enhanced Transport of 14C-Labeled Metabolites in Rice Seedlings in Relation to Ethylene-Stimulated Coleoptile Growth

To examine the effects of ethylene on sugar transport from endospermsto coleoptiles in rice (Oryza sativa L. cv. Sasanishiki) seedlings,the contents of free sugars in the coleoptiles of explants fedcold glucose and the distributions of ¹⁴C-activities after feedingof ¹⁴C-glucose to the scutella were determined at various timesafter ethylene application. Changes in sucrose, glucose andfructose in the cold glucose-fed explants exposed to ethylenewere similar to those in the ethylene-treated intact seedlingshaving endosperms. Ethylene enhanced the transport of ¹⁴C-labeledmetabolites from the scutella to the coleoptiles. Most of the¹⁴C accumulated in the ethylene-treated coleoptiles were presentas neutral substances in the ethanol-soluble fraction. Regardlessof the presence or absence of ethylene, the incorporation of¹⁴C into sucrose preceded that into glucose and fructose. Theglucose and fructose moieties of ¹⁴C-sucrose in the coleoptileswere almost equally labeled, and the specific activities of¹⁴C-sucrose were higher than those of ¹⁴C-glucose and ¹⁴C-fructose.These results suggested that sucrose synthesized in the scutellawas exported to the coleoptiles, and cleaved there into glucoseand fructose. Ethylene may accelerate the transport of ¹⁴C-labeledmetabolites by activating sucrose cleavage in the coleoptiles. (Received July 1, 1985; Accepted September 17, 1985)     

Aldehyde Reactions in Tissues in Relation to the Feulgen Technic

The nature of the Schiff-positive radical responsible for the Feulgen reaction was studied by several standard tests for aldehyde. Oxidative, amine, alkali, and catalytic reactions for aldehydes were used. An aldehyde reaction, similar histologically to the Feulgen, could not be produced by the oxidative and amine technics. However, by means of alkali and catalytic methods, it was possible to block the Feulgen-positive tissue radicals ordinarily released by acid hydrolysis. A second hydrolysis of the tissues (after the blocking reactions) restored most of their original Feulgen-positive characteristics.     

Sucrose Concentration Gradients along the Post-Phloem Transport Pathway in the Maternal Tissues of Developing Wheat Grains

Sucrose concentrations were measured in serial frozen sections of the post-phloem transport pathway in developing wheat (Triticum aestivum L.) grains. In normally importing grains, there was an approximately linear concentration gradient along the pathway, with a difference between the ends of the pathway of about 180 mM. This indicates an unusually low resistance for cell-to-cell transport, due perhaps to the large size-exclusion limit for the pathway. However, the existence of concentration gradients raises presently unresolvable questions about the relative contributions of diffusion versus bulk flow to transport within the symplast. The concentration gradient disappeared when sucrose movement ceased (i.e. in excised grains or when endosperm cavities of attached grains were perfused with p-chloromercuribenzene sulfonate [PCMBS] or with 1660 mOsm sorbitol). PCMBS appeared to block solute release into the endosperm cavity, whereas the sorbitol treatment, previously shown to cause localized plasmolysis in the chalaza, appeared to block movement across the chalaza. Sieve element/companion cell unloading appears to be an important control point for assimilate import. The sucrose concentration gradient and, probably, turgor and osmotic gradients are extremely steep there. PCMBS blocked import without affecting the sucrose concentration in the vascular parenchyma around the phloem. Thus, blockage of unloading was more complex than a simple "backing up" of solutes in the vascular parenchyma.     

Preferential Delivery of Zinc to Developing Tissues in Rice Is Mediated by P-Type Heavy Metal ATPase OsHMA2

Developing tissues such as meristems and reproductive organs require high zinc, but the molecular mechanisms of how zinc taken up by the roots is preferentially delivered to these tissues with low transpiration are unknown. Here, we report that rice (Oryza sativa) heavy metal ATPase2 (OsHMA2), a member of P-type ATPases, is involved in preferential delivery of zinc to the developing tissues in rice. OsHMA2 was mainly expressed in the mature zone of the roots at the vegetative stage, but higher expression was also found in the nodes at the reproductive stage. The expression was unaffected by either zinc deficiency or zinc excess. OsHMA2 was localized at the pericycle of the roots and at the phloem of enlarged and diffuse vascular bundles in the nodes. Heterologous expression of OsHMA2 in yeast (Saccharomyces cerevisiae) showed influx transport activity for zinc as well as cadmium. Two independent Tos17 insertion lines showed decreased zinc concentration in the crown root tips, decreased concentration of zinc and cadmium in the upper nodes and reproductive organs compared with wild-type rice. Furthermore, a short-term labeling experiment with ⁶⁷Zn showed that the distribution of zinc to the panicle and uppermost node I was decreased, but that, to the lower nodes, was increased in the two mutants. Taken together, OsHMA2 in the nodes plays an important role in preferential distribution of zinc as well as cadmium through the phloem to the developing tissues.Zinc is an essential metal for all organisms. In human genome, about 2,800 proteins, which account for 10% of total proteins, require zinc for structural or functional activities (Andreini et al., 2009; Maret and Li, 2009). In plants, zinc is involved in many enzyme activities, maintenance of integrity of biomembranes, RNA and DNA metabolism, carbohydrate metabolism, cell division, protein synthesis, gene expression regulation, and so on (Broadley et al., 2011). Zinc is also required for the metabolism of auxin. Therefore, zinc deficiency in plants causes stunted growth and “little leaf” (Broadley et al., 2011).Developing tissues with low transpiration especially requires high zinc for the active cell division and growth. For example, in pollen tubes, the zinc concentration at the growing tip was about 150 µg g^–1 dry weight compared with about 50 µg g^–1 in more basal regions (Ender et al., 1983). In the newly emerged root tips of wheat (Triticum aestivum) plants, zinc concentration is about 220 µg g^–1 (Ozturk et al., 2006). In rice (Oryza sativa) shoot meristems, more than 10 times the amount of zinc was found compared with mature leaf blades (Kitagishi and Obata, 1986). However, the molecular mechanisms underlying preferential delivery of zinc to these developing tissues are unknown.Zinc is taken up by the roots through the ZIP (for ZRT, IRT-related proteins) transporters (Grotz et al., 1998; Guerinot, 2000). Release of zinc into the xylem (xylem loading) is mediated by two P1B-type ATPases, AtHMA2 and AtHMA4, in Arabidopsis (Arabidopsis thaliana), which are localized at the pericycle (Hussain et al., 2004; Verret et al., 2004; Wong and Cobbett, 2009; Wong et al., 2009). Knockout of AtHMA4 and AtHMA2 or AtHMA4 alone resulted in a reduced translocation of zinc from the roots to the shoots. Recently, mutation of OsHMA2 was also reported to cause decreased translocation of zinc and cadmium from the roots to the shoots at the vegetative growth stage in rice (Satoh-Nagasawa et al., 2012; Takahashi et al., 2012); however, the exact mechanism underlying the involvement of this gene in the root-shoot translocation is not yet elucidated. Different from other metals such as manganese, zinc taken up by the roots is preferentially translocated to the shoot meristems and other developing tissues to meet the high demand of zinc in these tissues. In rice, ⁶⁵Zn taken up by the roots is translocated not to the expanded and active leaves with high transpiration but to the developing tissues, including unexpanded leaf blade, leaf sheath, and young panicles with very low transpiration within 1 h (Obata et al., 1980; Obata and Kitagishi, 1980a). By contrast, manganese taken up by the roots is translocated to the expanded and active leaves (Obata and Kitagishi, 1980a). Higher and faster zinc accumulation was observed at the basal part of elongating leaf sheath, which includes intercalary meristem (Obata et al., 1980; Obata and Kitagishi, 1980a; Kitagishi and Obata, 1986). High zinc accumulation in the meristem was also reported in tomato (Solanum lycopersicum; Langston, 1956) and subterranean clover (Trifolium subterraneum; Riceman and Jones, 1958a, 1958b). These observations suggest that distribution of zinc to the developing tissues does not depend on transpiration-dependent xylem-mediated transport.At the reproductive growth stage, the preferential accumulation of zinc was also found in the nodes of rice, which probably act as relay points for zinc distribution to the panicles (Obata and Kitagishi, 1980b), which have high zinc requirement for filling the grain. Deficiency of zinc causes low fertility (Obata and Kitagishi, 1980a). However, the molecular mechanisms on how zinc is preferentially delivered to these developing tissues have not been understood. In this study, we found that a member of P1B-type ATPase, OsHMA2, is involved in this process. OsHMA2 was previously implicated in the root-to-shoot translocation of zinc and cadmium at the vegetative stage (Satoh-Nagasawa et al., 2012; Takahashi et al., 2012). However, we found that the major role of OsHMA2 in the nodes is to preferentially deliver zinc to the developing tissues. This is especially important for delivering zinc to the grain at the reproductive growth stage. We also found that this transporter is responsible for cadmium distribution to the grains in rice.     

Auxin Transport in Secondary Tissues1

Auxin transport was investigated in excised stem segments ofNicotiana tabacum L. by the agar block technique using [1-¹⁴C]indol-3yl-acetic acid (IAA). The ability of the stems to transportauxin basipetally increased as secondary development proceeded;by contrast the ability of the pith to transport auxin declinedwith age. By separation of the stem tissues it was shown thatthe great majority of auxin transport took place in cells associatedwith the internal phloem and in cells close to the cambium;in both cases similar velocities of transport were found (c.5.0 mm h^–1 at 22°C). The effects of osmotic gradientson auxin transport through the internal phloem were investigated.IAA was found by chromatography to account for practically allthe radioactivity in receiver blocks and other extracts of stemsegments. The significance of these results is discussed.     

多胚苗水稻APIV不同颖花的多卵和多胚苗频率研究

APIV的多卵和多胚苗频率与其颖花和种子的发育季节以及颖花的着生部位有一定关系。在夏季条件下抽穗开花,其多卵频率和多胚苗频率均比较低,而在秋季条件下抽穗开花则比较高。在同一稻穗中的多卵和多胚苗频率,强势颖花要明显地高于弱势颖花。在同—季节抽穗开花的主穗和分蘖穗之间,其颖花内的多卵频率不存在明显差异,多胚苗频率的差异也不明显。     

Structural Differentiation of the Nucellar Epidermis in the Caryopsis of Rice (Oryza sativa)

In the developing caryopsis of rice (Oryza sativa L.) the nucellarepidermis forms a uniseriate layer through which assimilatesare transported to the endosperm. An anatomical study demonstratedthat the nucellar epidermal cells are fusiform in shape andare hexagonally packed. The anticlinal walls of the nucellarepidermis are characterized by ribs of wall-thickening whichare orientated radially with respect to the caryopsis. The wall-thickeningsappear to be cellulosic primary walls, as indicated by theirstaining with Calcofluor and periodic acid-Schiff's reagent.It is proposed that the geometry of the nucellar cells and theribs of wall-thickening are structural adaptations to resistthe compressional force which is placed on the nucellar epidermisduring the latter stages of grain filling. Oryza sativa, rice, caryopsis, grain filling, nucellar epidermis, wall-thickening     

小麦颖果果皮绿色层内同化物的合成与分配

小麦幼嫩颖果中,果皮内侧发育出由内表皮与亚表皮组成的一薄层绿色组织。显微与亚显微结构观察表明,虽绿色层只接受到自然光照的1/4～1/8,叶绿体仍能正常发育,叶绿素含量与叶绿体数量均高出旗叶。大量胞间连丝联接相邻的绿色细胞,并在一定时期形成开放的胞间通道,显然有利于同化物的快速胞间运输。离体颖果饲喂~14CO_2试验证明,新合成的同化物从绿色细胞输向胚珠。绿色层产生的同化物可能通过合点端的珠心进入胚乳或通过珠孔直接汇聚到胚珠和分化中的原胚。     

Transport of Radioactivity in Relation to Bracts in the Cotton Plant

[¹⁴C]sucrose was applied to the leaf subtending 14-d-old boll(cotton fruit) with and without bracts, and to bracts alonein var. Suvin (Gossypium barbadense L.). The removal of bractssubstantially enhanced the transport of radioactivity from theleaf to the carpels and seed cotton. When bracts alone weretreated, still larger quantities of radioactivity were incorporatedin the carpels and seed cotton. The bracts appeared to nourishthe developing boll through their own photosynthate and regulatethe transport of assimilate from the leaf. Radioactivity, transport, bracts, boll, carpels, seed cotton     

Discontinuities in the Temperature Function of Transmembrane Water Transport in Chara: Relation to Ion Transport

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P _d ) were observed delimiting a region of low activation energy (E _a around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high E _a (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15–30°C and lipid matrix-mediated transport beyond this region. The K⁺ channel inhibitor, tetraethylammonium as well as the Cl⁻ channel inhibitor, ethacrynic acid, diminished P _d in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K⁺ transport in Chara, only increased P _d below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K⁺ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K⁺ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K⁺ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29°C. Received: 12 August 1998/Revised: 13 November 1998     

粒重差异显著的两种水稻颖果发育比较

为了探讨控制水稻(Oryza sativa L.)颖果发育的因素,选择了颖果干重有显著差异的Ootikara(大粒,36mg/粒)和Habataki(小粒,18 mg/粒)两个水稻品种,比较颖果重量、胚乳细胞数、果皮和胚乳的结构以及某些生理活性等变化.结果指出:与Habataki相比,Ootikara子房壁细胞和颖果的持续生长期长,最终颖果的胚乳细胞数目和每个细胞的平均干重大;Ootikara颖果的脱氢酶和H2O2酶活性、穗的呼吸速率、剑叶的绿色程度和光合速率等维持高水平的时间长;Ootikara子房背部维管束失去功能的时间也较迟.结果表明,大粒品种的库容大和生理活性期长是其颖果能显著增大的生理原因.     

Enzyme Changes in Relation to Cell Growth in Excised Root Tissues1

Serial sections 10 mm. in length taken from the tip towardsthe base of the bean root have been cultured on 2 per cent,sucrose. At various time-intervals, length, invertase, phosphatase,and protein content of the sections have been determined. Alterationsin the enzyme complement of the sections have been related togrowth and protein content. The relation of changes occurringin excised fragments to those in the intact root have been discussed.     

Ontogenetic Changes in the Transport of Indol-3yl-acetic Acid into Maize Roots from the Shoot and Caryopsis

The quantities of endogenous indol-3yl-acetic acid (IAA) in endosperms and scutella of 6-day-old maize seedlings (Zea mays L. cv Giant White Horsetooth) were determined by a fluorimetric method. Endosperms were found to contain 33.4 nanograms IAA per plant, and scutella 7.5 nanograms IAA per plant. [5-³H]IAA applied to endosperms of 6-day-old seedlings moved into the roots and radioactivity accumulated at the apex of the primary root within 8 hours. Two to 7-day-old seedlings were treated simultaneously with [5-³H]IAA in the endosperm and [2-¹⁴C] IAA on the shoot apex. The patterns of transport into the root were found to change during ontogeny: in successively older plants, transport from the shoot into the roots increased relative to transport from the endosperm into the roots. The auxin required for the growth of maize roots could, therefore, partially be contributed by the shoot and endosperm. Ontogenetic changes in the relative importance of these two supplies could be of significance for the integration of growth and development between shoot and root.     

Silica Deposition in the Grass Leaf in Relation to Transpiration and the Effect of Dinitrophenol

Characteristic opal phytolith (‘silica body’) formationwas demonstrated in detached leaves of Sieglingia decumbens(Heath Grass), cultured in 100 ppm dissolved silicon (silicondioxide), previously, the leaves were free from intracellulardeposits as a result of silica-minimal tiller growth. The formertechnique allowed the study of the leaf deposition processesindependently of apical and root tissues, under growth-cabinetand glasshouse conditions. Deposition in excised leaves wascompletely suppressed by a surface, monomolecular coating, thusindicating that total net water loss was a limiting factor,however, evaporation from the recipient, epidermal tissues perse was not a requirement for this in situ deposition Generally,apart from an apparent, cell site shift in one treatment, phytolith-formationwas unaffected by the presence of the metabolic inhibitor 2,4-dinitrophenol. Also, some evidence was obtained of the influxof germanium dioxide into epidermal ldioblasts, which indicateda non-specificity of the host cell for silica. These results and those of earlier studies suggest that passive,non-metabolic mechanisms could account for the transport, influx,and cell lumen polymerization of silica in the grass leaf.     

Cadmium Alters Mitochondrial Membrane Potential,Inhibits Electron Transport Chain Activity and Induces Callose Deposition in Rice Seedlings

Journal of Plant Growth Regulation - In the present study, seedlings of indica rice (Oryza sativa L.) cultivars were subjected to 100 and 200 µM Cd(NO3)2 treatment in hydroponics for...     

粒重差异显著的两种水稻颖果发育比较

为了探讨控制水稻(Oryza sativa L.)颖果发育的因素,选择了颖果干重有显著差异的Ootikara (大粒,36mg/粒)和Habataki (小粒,18 mg/粒)两个水稻品种,比较颖果重量、胚乳细胞数、果皮和胚乳的结构以及某些生理活性等变化。结果指出:与Habataki相比, Ootikara子房壁细胞和颖果的持续生长期长,最终颖果的胚乳细胞数目和每个细胞的平均干重大; Ootikara颖果的脱氢酶和H2O2酶活性、穗的呼吸速率、剑叶的绿色程度和光合速率等维持高水平的时间长;Ootikara子房背部维管束失去功能的时间也较迟。结果表明,大粒品种的库容大和生理活性期长是其颖果能显著增大的生理原因。     

Uptake and Transport of Fusicoccin in Higher Plant Tissues

The uptake and release of ³H-labelled fusicoccin (FC) fed topea internode segments and the transport of [³H]FC in wholeplants and in excised parts of plants of pea, maize, and pumpkinhave been investigated. FC uptake showed three phases apparentlycorresponding to the penetration of FC: (1) into the free space;(2) into the cytoplasm; (3) into the vacuole. The first phase is a little thermosensitive and apparently includesadsorption binding to some free space component (cell surface?).The second and third phases are highly temperaturesensitiveand in part energy-dependent. However, no accumulation of FCagainst a concentration gradient is observed at high externalFC concentration. Accumulation at low concentration seems dueto the formation of a poorly exchangeable complex with somecell structure. The efflux of the FC penetrated within the cells is limitedby some highly temperature-sensitive process. A small fractionof the accumulated FC tends to be retained in the tissue, eitherbecause sequestered in some compartment or because bound tosome cellular component. FC transport in whole plants as well as in isolated plant partsseems to depend mainly on simple diffusion and on transportby mass flow in the xylem. Long distance transport of the toxinin pumpkin plants mainly occurs in the xylem, while the slowmovement in non-vascular tissues seems to depend on diffusioncombined with mass flow in the free space, and does not seemheavily influenced by metabolic factors.