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PubMed1997

Study of lignification by noninvasive techniques in growing maize internodes. An investigation by Fourier transform infrared cross-polarization-magic angle spinning 13C-nuclear magnetic resonance spectroscopy and immunocytochemical transmission electron microscopy.

Noninvasive techniques were used for the study in situ of lignification in the maturing cell walls of the maize (Zea mays L.) stem. Within the longitudinal axis of a developing internode all of the stages of lignification can be found. The synthesis of the three types of lignins, p-hydroxyphenylpropane (H), guaiacyl (G), and syringyl (S), was investigated in situ by cross-polarization-magic angle spinning 13C-solid-state nuclear magnetic resonance, Fourier transform infrared spectroscopy, and immunocytochemical electron microscopy. The first lignin appearing in the parenchyma is of the G-type preceeding the incorporation of S nuclei in the later stages. However, in vascular bundles, typical absorption bands of S nuclei are visible in the Fourier transform infrared spectra at the earliest stage of lignification. Immunocytochemical determination of the three types of lignin in transmission electron microscopy was possible thanks to the use of antisera prepared against synthetic H, G, and the mixed GS dehydrogenative polymers (K. Ruel, O. Faix, J.P. Joseleau [1994] J Trace Microprobe Tech 12: 247-265). The specificity of the immunological probes demonstrated that there are differences in the relative temporal synthesis of the H, G, and GS lignins in the different tissues undergoing lignification. Considering the intermonomeric linkages predominating in the antigens used for the preparation of the immunological probes, the relative intensities of the labeling obtained provided, for the first time to our knowledge, information about the macromolecular nature of lignins (condensed versus noncondensed) in relation to their ultrastructural localization and development stage.

Animals↗
PubMed2000

The role of the Arabidopsis ELD1 gene in cell development and photomorphogenesis in darkness.

Because cell growth and differentiation are regulated by complex interactions among different signaling pathways, a growth defect affects subsequent differentiation. We report on a growth-defective mutant of Arabidopsis, called eld1 (elongation defective 1). Cell elongation was impaired in every organ examined. Later characteristics of the eld1 phenotype include defective vascular tissue differentiation, the inability to grow in soil, ectopic deposition of suberin around twisted vascular bundles, the de-etiolation phenotype, and continuation of shoot development and flowering in the dark. The dwarf phenotype of eld1 could not be rescued by treatment with exogenous growth regulators. Because defective cell elongation is the earliest and most universal feature detected in eld1 mutants, control of or activity in cell elongation may be the primary function of the ELD1 gene. The impaired cell growth results in pleiotropic effects on cell proliferation and differentiation, and the retardation in hypocotyl elongation enables growth and development in darkness.

Arabidopsis↗
PubMed2000

Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma.

The tomato (Lycopersicon esculentum cv Ailsa Craig) polygalacturonase genes TAPG1 (LYCes;Pga1;2) and TAPG4 (LYCes;Pga1;5) are abundantly expressed in both abscission zones and the pistils of mature flowers. To further investigate the spatial and temporal expression patterns for these genes, the TAPG gene promoters were ligated to beta-glucuronidase (GUS) reporter genes and transformed into tomato. GUS expression with both constructs was similar and entirely consistent with the expression patterns of the native gene transcripts. GUS activity was observed in the weakening abscission zones of the leaf petiole, flower and fruit pedicel, flower corolla, and fruit calyx. In leaf petiole and flower pedicel zones this activity was enhanced by ethylene and inhibited by indole-3-acetic acid. On induction of abscission with ethylene, GUS accumulation was much earlier in TAPG4:GUS than in TAPG1:GUS transformants. Moreover, TAPG4:GUS staining appeared to predominate in the vascular bundles relative to surrounding cortex cells whereas TAPG1:GUS was more evenly distributed across the separation layer. Like the native genes, GUS was also expressed in the stigma. Activity was not apparent in pistils until the flowers had opened and was confined to the stigma and style immediately proximal to it. A minimal promoter construct consisting of a 247-bp 5'-upstream element from TAPG1 was found to be sufficient to direct GUS expression in both abscission zones and the stigma.

Ethylenes↗
PubMed2000

Sugar uptake and transport in rice embryo. Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar and light.

We investigated sugar uptake and transport in rice (Oryza sativa) embryo during grain germination. Endogenous sugar levels, accumulation of starch granules, and gene expression of a rice sucrose transporter (OsSUT1) were examined using rice embryos germinated with or without exogenous sugar supply. Starch granules remarkably accumulated in the cells around vascular bundles as a consequence of the sugar taken up by the embryos, indicating that the taken-up sugars are transiently converted into starch. In situ detection for OsSUT1 mRNA indicated its localization in the phloem companion cells. Furthermore, northern-blot and in situ hybridization analyses showed that OsSUT1 expression is not detectable in embryos subjected to sugar starvation conditions, whereas its expression is enhanced by an increased endogenous sugar level. Overall results indicate that the expression of companion cell-specific sucrose transporter, OsSUT1 is regulated by the endogenous sugar status as well as light exposure.

Biological Transport↗
PubMed2000

Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis.

Profilin (PFN) is an ubiquitous, low-M(r), actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.

Actins↗
PubMed2001

Evidence for symplastic phloem unloading in sink leaves of barley.

The pathway of phloem unloading in sink barley (Hordeum vulgare) leaves was studied using a combination of electron microscopy, carboxyfluorescein transport, and systemic movement of barley stripe mosaic virus expressing the green fluorescent protein. Studies of plasmodesmatal frequencies between the phloem and mesophyll indicated a symplastic sieve element- (SE) unloading pathway involving thick-walled and thin-walled SEs. Phloem-translocated carboxyfluorescein was unloaded rapidly from major longitudinal veins and entered the mesophyll cells of sink leaves. Unloading was "patchy" along the length of a vein, indicating that sieve element unloading may be discontinuous along a single vascular bundle. This pattern was mirrored precisely by the unloading of barley stripe mosaic virus expressing the green fluorescent protein. Transverse veins were not utilized in the unloading process. The data collectively indicate a symplastic mechanism of SE unloading in the sink barley leaf.

Biological Transport↗
PubMed2001

Developmental and stress regulation of RCI2A and RCI2B, two cold-inducible genes of arabidopsis encoding highly conserved hydrophobic proteins.

The capability of most higher plants to tolerate environmental conditions strongly depends on their developmental stage. In addition, environmental factors have pleiotropic effects on many developmental processes. The interaction between plant development and environmental conditions implies that some genes must be regulated by both environmental factors and developmental cues. To understand their developmental regulation and obtain possible clues on their functions, we have isolated genomic clones for RCI2A and RCI2B, two genes from Arabidopsis ecotype Columbia (Col), whose expression is induced in response to low temperature, dehydration, salt stress, and abscisic acid. The promoters of RCI2A and RCI2B were fused to the uidA (GUS)-coding sequence and the resulting constructs used to transform Arabidopsis. GUS activity was analyzed in transgenic plants during development under both stressed and unstressed conditions. Transgenic plants with either the RCI2A or RCI2B promoter showed strong GUS expression during the first stages of seed development and germination, in vascular bundles, pollen, and most interestingly in guard cells. When transgenic plants were exposed to low temperature, dehydration, salt stress, or abscisic acid, reporter gene expression was induced in most tissues. These results indicate that RCI2A and RCI2B are regulated at transcriptional level during plant development and in response to different environmental stimuli and treatments. The potential role of RCI2A and RCI2B in plant development and stress response is discussed.

Abscisic Acid↗
PubMed1966

Physiological studies on pea tendrils. I. Growth and coiling following mechanical stimulation.

Unbranched tendrils arising from the fifth node of light grown Alaska pea plants were found to reach maturity at the age of 10 days. Such tendrils, when stroked, coiled rapidly. They remained maximally irritable for at least 3 days.Coiling was separable into 2 components, curvature (measured in degrees) and elongation. Coiling reached a maximum from one-half to 3 hours after tactile stimulation. This maximum is followed by a decrease, then by a further increase up to 48 hours.The optimum temperature range for curvature was 16 to 30 degrees after 2 hours and 16 to 20 degrees after 20 hours. Curvature was minimal and elongation optimal at a pH of 6.6. Both curvature and elongation were greater in white light than in darkness.Severing the dorsal vascular bundles decreased coiling, whereas severing the ventral bundles had no effect. Amputation of the tip of an excised tendril increased curvature but decreased elongation. In some cases, coiling of tendrils in situ was followed by curvature of the subjacent stem.Various growth substances produce effects on elongation and curvature of tendrils in vitro, in light and dark. CCC and GA decrease curvature in the light and increase curvature in the dark and elongation in the light and in the dark. Kinetin decreases curvature in the light, increases it slightly in the dark and has no effect on elongation. IAA increases elongation in the light and in the dark at concentrations above 10(-6)m. At lower concentrations, curvature was increased in the light and decreased in the dark.These data are discussed with a view towards explaining the coiling event.

Journal Article↗
PubMed1969

Phosphorus compounds in translocating Phloem.

Phosphate-(32)P was introduced into a turnip leaf, and 3 hr later, the vascular bundles were stripped from the petiole and their phosphate ester pattern was studied. The pattern did not alter along their length and was like that of other tissues. Pumpkin leaves were painted with phosphate-(32)P; and later, the petioles were cut, the sieve tube exudates were collected and their phosphate ester patterns were studied. Exudates collected after 10 min had a high proportion of their (32)P present in P(i) and nucleoside triphosphates, while exudates collected after long translocation times (4-22 hr) had a lower proportion in these, and a higher proportion in hexose monophosphates and UDP glucose. In general, the ester patterns were like those of other tissues. The results indicate that sieve tubes are metabolically active, and that P(i) is the primary form in which phosphorus moves in the phloem.

Journal Article↗
PubMed1975

Movement of C-labeled Sugars into Kernels of Wheat (Triticum aestivum L.).

An anatomical study of wheat (Triticum aestivum L.) kernels 14 days after anthesis revealed that the tracheary elements of the pericarp vascular bundle are not in direct continuity with those of the rachilla. The phloem was continuous from the rachilla into the crease of the pericarp.Shortly after exposure of the flag leaf to (14)CO(2), relatively high proportion of the (14)C extracted from the pericarp and endosperm was found in glucose and fructose. With additional time, the percentage of (14)C in the monosaccharides declined and that in sucrose increased to a maximum 3 hours after (14)CO(2) exposure. The monosaccharides comprised about one-half of the soluble sugars extracted from the pericarp. Based on these observations, it appeared that sucrose hydrolysis might be prerequisite to sugar movement from the terminal phloem elements in the pericarp and into the endosperm. However, when (14)C-fructosyl-sucrose was injected into the peduncle, there was little additional randomization of the (14)C between the glucose and fructose moieties of sucrose extracted from the pericarp and endosperm compared to the rachis sucrose. If we assume that injected sucose was transported to the kernels via the phloem, then either sucrose moves out of the terminal phloem elements in the pericarp and into the endosperm unaltered, or if hydrolysis and resynthesis are a prerequisite to transport into the endosperm, the products of hydrolysis are not freely available for isomerization.

Journal Article↗
PubMed1979

Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest.

Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.Protease activity increased in nodules from harvested plants, reached a maximum at day 7 after harvest, and then declined to a level almost equal to the control by day 22 after harvest. Soluble protein and leghemoglobin decreased in nodules from harvested plants in an inverse relationship to protease activity.Nitrate reductase activity of nodules from harvested plants increased significantly within 24 hours and was inversely associated with acetylene reduction. The difference in nitrate reductase between nodules from harvested plants and control plants became less evident as shoot regrowth occurred and as acetylene reduction increased in the harvested plants.No massive loss of nodules occurred after harvest as evidenced by little net change in nodule fresh weight. There was, however, a rapid localized senescence which occurred in nodules of harvested plants. Histology of nodules from harvested plants showed that they degenerated at the proximal end after harvest. Starch in the nodule was depleted by 10 days after harvest. The meristem and vascular bundles of nodules from harvested plants remained intact. The senescent nodules began to regrow and fix nitrogen after shoot growth resumed.

Journal Article↗
PubMed1982

Sucrose and Glucose Uptake into Beta vulgaris Leaf Tissues : A Case for General (Apoplastic) Retrieval Systems.

Concentration curves for sugar and amino acid uptake by Beta vulgaris L. leaf tissues contained both a saturable and a linear component. Similarly shaped curves were obtained for influx of sucrose, glucose, and 3-O-methyl glucose by leaf discs, whole petiole slices, petiole segments containing pith tissue only, and petiole segments containing vascular bundles, although the tissues took up the various sugars via different proportions of saturable versus linear uptake. Two millimolar p-chloromercuribenzenesulfonic acid selectively inhibited the saturable component of sucrose uptake, but had almost no effect on the linear component. Uptake of glucose and 3-O-methyl glucose remained unaffected by p-chloromercuribenzenesulfonic acid treatment. Anoxia was found to inhibit the linear component of both sucrose and 3-O-methyl glucose influx, while the saturable component remained unaffected. The linear component of sucrose uptake was also competitively inhibited by maltose, as well as being selectively promoted by certain exposures to 5 millimolar N-ethylmaleimide, 2 micrograms per milliliter cycloheximide, and high levels of mannitol acting as osmoticum. These results support the proposal that the linear component is due to a process more complex than simple, or exchange, diffusion. It would also appear that the linear transport component utilizes a separate energy source than does the saturable component of sucrose influx.Evidence for phloem loading from the apoplast was re-examined with respect to the present findings. Saturable sucrose uptake by minor vein tissues may represent retrieval of solute from the free space, which could explain the ;apoplastic loading' phenomenon.

Journal Article↗
PubMed1984

Regulatory effect of cytokinin on secondary xylem fiber formation in an in vivo system.

The regulatory effect of cytokinin on the formation of secondary xylem fibers was studied in the hypocotyl of young Helianthus annuus L. plants. Positive correlation was found between the kinetin supplied (0.25-0.5 micrograms/gram) to the growth medium and the rate of fiber formation within and between the vascular bundles. Reducing the root originated cytokinin supply, either by root removal or by lowering the transpiration rate, diminished the number of newly formed secondary xylem fibers. This decrease was considerably reversed in the presence of 0.5 microgram/gram kinetin. Early pulse exposure of kinetin had a temporary promoting effect on fiber differentiation at low concentrations and a permanent inhibitory effect at high concentration.

Journal Article↗
PubMed1984

Intracellular localization of lipoxygenases-1 and -2 in germinating soybean seeds by indirect labeling with protein a-colloidal gold complexes.

Soybean lipoxygenases-1 and -2 were localized intracellularly in seeds at various stages of germination by indirect labeling of cryosections with protein A-colloidal gold complexes. Two sizes of gold particles (Au(5) and Au(16)) were used in single- and double-labeling experiments. In primary leaves, lipoxygenases are demonstrated to occur in vacuolating parenchyma cells but not in massive, nondifferentiated cells. In cotyledons, both isoenzymes are localized in the cytoplasm of storage parenchyma cells and in an aberrant type of protein bodies, occurring in hypodermis and vascular bundle sheath cells. No association has been found with either protein bodies in storage parenchyma cells or lipid bodies, mitochondria, and other organelles in any type of cell. The possible significance of lipoxygenase in the metabolism of storage lipids and its possible function as a regulatory enzyme are discussed on the basis of the random distribution throughout the cytoplasm of storage parenchyma cells and the course of biochemical processes during seed germination.

Journal Article↗
PubMed1985

Permeability of the suberized mestome sheath in winter rye.

Mestome sheath cells of winter rye (Secale cereale L. cv Puma) deposit suberized lamellae in their secondary cell walls. Histochemical tests including acid digestion and staining with Sudan IV and Chelidonium majus root extract were used to detect the presence of suberin in the primary cell wall. There was no evidence of a Casparian band between adjacent mestome sheath cells. Fluorescent dye techniques were used to trace solute movement through the rye leaf apoplast. Calcofluor white M2R, a fluorescent dye which binds to cell walls as it moves apoplastically, proved to be too limited in its mobility in leaves to test mestome sheath permeability. Trisodium 3-hydroxy-5,8,10 pyrene trisulfonate, a fluorescent dye which is mobile in the apoplast, moved easily up the vascular bundles in the transpiration stream, and diffused outward from the veins to the epidermal cell walls within minutes of reaching a particular level in the leaf. We conclude that the suberized mestome sheath of rye leaves is freely permeable to solutes moving apoplastically through radial primary cell walls.

Journal Article↗
PubMed1988

The relation of anatomy to water movement and cellular response in young barley leaves.

Young barley (Hordeum vulgare L. cv Arivat) leaves were examined anatomically and physiologically to infer the pathway of transpirational water movement and to explain why the growing region is more responsive to osmotic stress than the expanded blade. Vessels with open lumens extend from the intercalary meristem to the expanded blade, and all vessels are clustered in five vascular bundles that are separated by 20 closely packed mesophyll cells. Heat pulse transport data confirmed the anatomical suggestion that water moves through the growing region in vessels and not intercellularly, and also showed that stress reduces xylem water transport within 1 minute while transpiration remained unaffected. Water equal in volume to twice that expected in the xylem, and which exchanges more readily with water in the nutrient solution than with most water in tissues, can be extracted easily from growing tissues. It is hypothesized that this water is xylem plus cell wall water, that osmotic stress will quickly reduce its in situ water potential, and that stress causes growth to stop because cells in the growing region can respond rapidly to changes in water potential around them. In the expanded blade, bundles containing vessels are three and eight cells away from the closest and next substomatal cavities. This allows xylem water loss to occur predominantly through the closest stomata, and the expanded blade is believed to be less responsive because effects of stress on xylem water potential are confined largely to cells immediately around the vessels.

Journal Article↗
PubMed1989

Localization and pattern of graviresponse across the pulvinus of barley Hordeum vulgare.

Pulvini of excised stem segments from barley (Hordeum vulgare cv Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0 degrees and the lowermost point 180 degrees. A gravity-induced increase in pulvinus thickness was observable within 40 degrees of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.

Cell Division↗
PubMed1990

Postphloem, nonvascular transfer in citrus: kinetics, metabolism, and sugar gradients.

Postphloem, nonvascular assimilate transport occurs over an unusually long area in citrus fruit and thus facilitates investigation of this process relative to sugar entry into many sink structures. Labeled photosynthates moving into juice tissues of grapefruit (Citrus paradisi Macf.) slowed dramatically after entering the postphloem transport path (parenchyma cells, narrow portions of segment epidermis, and hair-like, parenchymatous stalks of juice sacs). Kinetic, metabolic, and compositional data indicated that transfer through the nonvascular area was delayed many hours by temporary storage and/or equilibration with sugars in compartments along the postphloem path. Labeled assimilates were generally recovered as sucrose throughout the path, and extent of hexose formation enroute bore no apparent relationship to the assimilate transfer process. Even after 24 hours, radiolabel was restricted to discrete, highly localized areas directly between vascular bundles and juice sacs. Postphloem transfer occurred against an ascending sucrose concentration gradient in young fruit, whereas a descending gradient (favoring diffusion/cytoplasmic streaming) developed only later in maturation. Involvement of a postphloem bulk flow is complicated in the present instance by the extremely limited water loss from juice sacs either via transpiration or fluid backflow. Nonetheless, tissue expansion can account for a collective water inflow of at least 1.0 milliliter per day throughout the majority of juice sac development, thus providing a modest, but potentially important means of nonvascular solution flow. Overall, data indicate postphloem transfer (a) can follow highly localized paths through sizable nonvascular areas (up to 3.0 centimeters total), (b) appears to involve temporary storage and/or equilibration with compartmentalized sugars enroute, (c) can occur either against an overall up-hill sugar gradient (young tissues) or along a descending gradient (near full expansion), and (d) appears to involve at least some contribution by nonvascular mass flow accommodated by tissue expansion.

Journal Article↗