Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity

Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near −40 °C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree ( Cercidiphyllum japonicum ), of which XPCs exhibited deep supercooling to −40 °C, were prepared by methanol extraction. The crude extracts were purified by liquid–liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8–9.0 °C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3- O - β -glucoside (Q3G), kaempferol-7- O - β -glucoside (K7G), 8-methoxykaempferol-3- O - β -glucoside (8MK3G) and kaempferol-3- O - β -glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.     

DEVELOPMENT OF THE AMORPHOUS LAYER (PROTECTIVE LAYER) IN XYLEM PARENCHYMA OF CV. GOLDEN DELICIOUS APPLE,CV. LORING PEACH,AND WILLOW

The amorphous layer (AL) in xylem parenchyma may play a prominent role in defining the freezing response of a tissue. A comparative study was undertaken to examine the development of the AL in xylem parenchyma of cv. Golden Delicious apple and cv. Loring peach, which exhibit deep supercooling, and willow, which exhibits only extracellular freezing. AL formation did not begin until secondary wall formation was entirely completed. Deposition of the loose textured AL began at the edges of a pit cavity and spread to the adjacent vessel-parenchyma pit membrane. Upon completion, the AL ensheathed the protoplast of the xylem parenchyma cell and was much thicker in the area of the pit cavity. During AL development, numerous cortical microtubules, RER, and coated vesicles were present in the cytoplasm. Observations indicate that AL development was similar in all 3 species, but differences were found in the time of maturation within an annual ring and AL coloration after staining with toluidine blue O. The latter indicates that compositional differences may exist which may account for the variation in freezing response exhibited by these species.     

CHANGES IN THE ULTRASTRUCTURE OF XYLEM PARENCHYMA CELLS OF PEACH (PRUNUS PERSICA) AND RED OAK (QUERCUS RUBRA) IN RESPONSE TO A FREEZING STRESS

An ultrastructural investigation was conducted of xylem parenchyma cells of peach (Prunus persica [L.] Batsch.) cv. Harbrite and red oak (Quercus rubra L.) in response to a freezing stress. Freezing curves of xylem tissues, as determined by differential thermal analysis, were used to predict temperatures at which both living and dead cells would be observed. Tissues were exposed to low temperatures (-15 to -35 C) and fixed in a frozen state at -10C and at thawing. Current models of the freezing behavior of supercooled plant cells suggest that xylem parenchyma cells behave as individual water droplets. This implies that cells are unresponsive to the presence of low temperature and extracellular ice until internal nucleation triggers lethal, intracellular freezing. For these reasons, deep supercooling has been described as an avoidance mechanism. Results of this study confirmed earlier reports that xylem parenchyma cells freeze as individuals or in small groups. Individual cells, however, did not exhibit a neutral response. Instead, a range of responses was observed that included internal and external vesiculation, deep invaginations of the plasma membrane, and the formation of electron-dense deposits external to the plasmalemma. In general, our observations suggested that the cells responded to a dehydrative stress. Results are discussed in context of the biophysical data associated with deep supercooling phenomena and compared to responses of cells that exhibit extracellular freezing.     

Supercooling of xylem ray parenchyma cells in tropical and subtropical hardwood species

 The freezing behavior of xylem ray parenchyma cells in several woody species, Ficus elastica, F. microcarpa, Mangifera indica, Hibiscus Rosa-sinensis, and Schefflera arboricola, that are native to non-frost tropical and subtropical zones, was investigated by differential thermal analysis (DTA), cryo-scanning electron microscopy (cryo-SEM) and freeze-replica electron microscopy. Although profiles after DTA did not exhibit clear evidence of supercooling in the xylem ray parenchyma cells, electron microscopy revealed that the majority of xylem ray parenchyma cells in all of the woody species examined were supercooled to around –10°C upon freezing temperatures and were not frozen extracellularly. It seems likely that DTA failed to reveal the low temperature exotherm (LTE), that is produced by breakdown of supercooling in the xylem ray parenchyma cells as a consequence of the overlap between the high temperature exotherm and the LTE in each case. The xylem ray parenchyma cells in these woody species were very sensitive to dehydration, and supercooling had, to some extent, a protective effect against freezing injury. It is suggested that the capacity for supercooling of xylem ray parenchyma cells of tropical and subtropical woody species might be the result of inherent structural characteristics, such as rigid cell walls and compact xylem tissues, rather than the result of positive adaptation to freezing temperatures. The present and previous results together indicate that the responses of xylem ray parenchyma cells in a wide variety of hardwood species to freezing temperatures can be explained as a continuum, the specifics of which depend upon the temperatures of the growing conditions. Received: 24 January 1997 / Accepted: 13 May 1997     

Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi)

Xylem parenchyma cells (XPCs) in larch adapt to subfreezing temperatures by deep supercooling, while cortical parenchyma cells (CPCs) undergo extracellular freezing. The temperature limits of supercooling in XPCs changed seasonally from -30 degrees C during summer to -60 degrees C during winter as measured by freezing resistance. Artificial deacclimation of larch twigs collected in winter reduced the supercooling capability from -60 degrees C to -30 degrees C. As an approach to clarify the mechanisms underlying the change in supercooling capability of larch XPCs, genes expressed in association with increased supercooling capability were examined. By differential screening and differential display analysis, 30 genes were found to be expressed in association with increased supercooling capability in XPCs. These 30 genes were categorized into several groups according to their functions: signal transduction factors, metabolic enzymes, late embryogenesis abundant proteins, heat shock proteins, protein synthesis and chromatin constructed proteins, defence response proteins, membrane transporters, metal-binding proteins, and functionally unknown proteins. All of these genes were expressed most abundantly during winter, and their expression was reduced or disappeared during summer. The expression of all of the genes was significantly reduced or disappeared with deacclimation of winter twigs. Interestingly, all but one of the genes were expressed more abundantly in the xylem than in the cortex. Eleven of the 30 genes were thought to be novel cold-induced genes. The results suggest that change in the supercooling capability of XPCs is associated with expression of genes, including genes whose functions have not been identified, and also indicate that gene products that have been thought to play a role in dehydration tolerance by extracellular freezing also have a function by deep supercooling.     

The use of lanthanum to characterize cell wall permeability in relation to deep supercooling and extracellular freezing in woody plants: II. Intrageneric comparisons betweenBetula lenta andBetula papyrifera

Summary The permeability and porosity of xylem cell walls are believed to play a major role in defining the ability of a cell or tissue to exhibit deep supercooling. Lanthanum nitrate, was utilized to contrast the permeability of stem tissues inB. lenta, which exhibits deep supercooling, withB. papyrifera, which exhibits equilibrium freezing. Although the two species differed greatiy in their response to low temperature, distribution of lanthanum deposits was quite similar. Primary cell walls of all xylem cell types appeared permeable although lanthanum deposition was patchy. Secondary cell walls of fiber cells were also permeable to lanthanum whereas the secondary wall of vessel elements and xylem parenchyma appeared impermeable to the lanthanum. Pit membranes, in all cell types and the protective layer in xylem parenchyma frequently exhibited deposits of lanthanum. Results of this study indicate that the porosity and permeability of the pit membrane, rather than the entire cell wall may determine the rate of water loss from xylem parenchyma to sites of extracellular ice. If differences exist between the species in the physical structure of these sites, they may explain differences observed in their response to freezing.Abbreviations DTA differential thermal analysis - HTE high temperature exotherm - LTE low temperature exoterm - F fiber cell - V vessel element     

Ultrastructural Evidence That Intracellular Ice Formation and Possibly Cavitation Are the Sources of Freezing Injury in Supercooling Wood Tissue of Cornus florida L

Although cellular injury in some woody plants has been correlated with freezing of supercooled water, there is no direct evidence that intracellular ice formation is responsible for the injury. In this study we tested the hypothesis that injury to xylem ray parenchyma cells in supercooling tissues is caused by intracellular ice formation. The ultrastructure of freezing-stress response in xylem ray parenchyma cells of flowering dogwood (Cornus florida L.) was determined in tissue prepared by freeze substitution. Wood tissue was collected in the winter, spring, and summer of 1992. Specimens were cooled from 0 to -60[deg]C at a rate of 5[deg]C h-1. Freezing stress did not affect the structural organization of wood tissue, but xylem ray parenchyma cells suffered severe injury in the form of intracellular ice crystals. The temperatures at which the ice crystals were first observed depended on the season in which the tissue was collected. Intracellular ice formation was observed at -20, -10, and -5[deg]C in winter, spring, and summer, respectively. Another type of freezing injury was manifested by fragmented protoplasm with indistinguishable plasma membranes and damaged cell ultrastructure but no evidence of intracellular ice. Intracellular cavitation may be a source of freezing injury in xylem ray parenchyma cells of flowering dogwood.     

Effect of Macerase, Oxalic Acid, and EGTA on Deep Supercooling and Pit Membrane Structure of Xylem Parenchyma of Peach

The object of this study was to determine if calcium cross-linking of pectin in the pit membrane of xylem parenchyma restricts water movement which results in deep supercooling. Current year shoots of `Loring' peach (Prunus persica) were infiltrated with oxalic acid or EGTA solutions for 24 or 48 hours and then either prepared for ultrastructural analysis or subjected to differential thermal analysis. The effect of 0.25 to 1.0% pectinase (weight/volume) on deep supercooling was also investigated. The use of 5 to 50 millimolar oxalic acid and pectinase resulted in a significant reduction (flattening) of the low temperature exotherm and a distinct swelling and partial degradation of the pit membrane. EGTA (10 millimolar) for 24 or 48 hours shifted the low temperature exotherm to warmer temperatures and effected the outermost layer of the pit membrane. A hypothesis is presented on pectin-mediated regulation of deep supercooling of xylem parenchyma.     

Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell walls

Summary By cryo-scanning electron microscopy we examined the effects of the organization of the cell walls of xylem ray parenchyma cells on freezing behavior, namely, the capacity for supercooling and extracellular freezing, in various softwood species. Distinct differences in organization of the cell wall were associated with differences in freezing behavior. Xylem ray parenchyma cells with thin, unlignified primary walls in the entire region (all cells inSciadopitys verticillata and immature cells inPinus densiflora) or in most of the region (mature cells inP. densiflora and all cells inP. pariflora var.pentaphylla) responded to freezing conditions by extracellular freezing, whereas xylem ray parenchyma cells with thick, lignified primary walls (all cells inCrytomeria japonica) or secondary walls (all cells inLarix leptolepis) in most regions responded to freezing by supercooling. The freezing behavior of xylem ray parenchyma cells inL. leptolepis changed seasonally from supercooling in summer to extracellular freezing in winter, even though no detectable changes in the organization of cell walls were apparent. These results in the examined softwood species indicate that freezing behavior of xylem ray parenchyma cells changes in parallel not only with clear differences in the organization of cell walls but also with subtle sub-electron-microscopic differences, probably, in the structure of the cell wall.     

Seasonal changes in the freezing behavior of xylem ray parenchyma cells in four boreal hardwood species

The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely, Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis, was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates. Copyright 1999 Academic Press.     

Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

Sul trasporto dei glucidi nelle piante superiori: I. — Aspetti biochimici

Abstract

CARBOHYDRATE TRANSLOCATION IN HIGHER PLANTS. I. - BIOCHEMICAL ASPECTS. — The concentration of soluble sugars and of hexose phosphates and the activity of several enzymes involved in hexose activation and polysaccaride synthesis have been investigated, separately, in the phloematic tissue and in the medullar parenchyma of Cucurbita Pepo internodes.

In the phloematic tissue (including sieve tubes, companion cells and phloematic parenchyma) the concentration of free hexoses appeared of about 50% lower, and that of glucose-6-P and of sucrose of about 100% higher then in the medullar parenchyma. Consistent amounts of raffinose were found only in the phloematic tissue. Paper chromatograms of the sieve tube exudate showed the presence of raffinose and sucrose in a ratio close to unity, and no appreciable amounts of free hexoses.

Determination of enzyme activity on preparations obtained from homogenates from the two types of tissue by repeated ammonium sulfate precipitation showed in the phloematic tissue a high activity of the enzymes hexokinase, UDP-kinase, UDPG-pyrophosphorylase and inorganic pyrophosphatase. The presence in the same tissue of galactosekinase, UDP-Gal-pyrophosphorylase and UDPG-epimerase was also ascertained.

On a protein basis, the activity of UDPG-pyrophosphorylase, inorganic pyrophosphatase and hexokinase appared about 3 times higher in the phloematic tissue than in the parenchyma; while this difference between the two tissues was not so marked for phosphofructokinase, and very small for other enzymes such as ATP-ase and phosphomono-esterase.

These results suggest that the very high activity, in the phloem cells neighbouring the sieve tubes, of the enzyme system catalyzing oligopolysaccaride synthesis could be an important component of the mechanism involved in the accumulation of oligopolysaccarides in the sieve tubes, and thus in sugar translocation. A scheme is proposed according to which the ATP and UTP energy would be utilized by the phloem cells to reach and to maintein a concentration of soluble sugars consistently higher than that prevailing in the contiguous tissues.     

Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

Presence of supercooling-facilitating (anti-ice nucleation) hydrolyzable tannins in deep supercooling xylem parenchyma cells in <Emphasis Type="Italic">Cercidiphyllum japonicum</Emphasis>

Xylem parenchyma cells (XPCs) in trees adapt to subzero temperatures by deep supercooling. Our previous study indicated the possibility of the presence of diverse kinds of supercooling-facilitating (SCF; anti-ice nucleation) substances in XPCs of katsura tree (Cercidiphyllum japonicum), all of which might have an important role in deep supercooling of XPCs. In the previous study, a few kinds of SCF flavonol glycosides were identified. Thus, in the present study, we tried to identify other kinds of SCF substances in XPCs of katsura tree. SCF substances were purified from xylem extracts by silica gel column chromatography and Sephadex LH-20 column chromatography. Then, four SCF substances isolated were identified by UV, mass and nuclear magnetic resonance analyses. The results showed that the four kinds of hydrolyzable gallotannins, 2,2′,5-tri-O-galloyl-α,β-d-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-β-d-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-β-d-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside (pentagalloyl Glc), in XPCs exhibited supercooling capabilities in the range of 1.5–4.5°C, at a concentration of 1 mg mL⁻¹. These SCF substances, including flavonol glycosides and hydrolyzable gallotannins, may contribute to the supercooling in XPCs of katsura tree.     

Stem anatomy of Dolichos lablab Linn (Fabaceae): Origin of cambium and reverse orientation of vascular bundles

Secondary growth in the stem of Dolichos lablab is achieved by the formation of eccentric successive rings of vascular bundles. The stem is composed of parenchymatous ground tissue and xylem and phloem confined to portions of small cambial segments. However, development of new cambial segments can be observed from the obliterating ray parenchyma, the outermost phloem parenchyma and the secondary cortical parenchyma. Initially cambium develops as small segments, which latter become joined to form a complete cylinder of vascular cambium. Each cambial ring is functionally divided into two distinct regions. The one segment of cambium produces thick-walled lignified xylem derivatives in centripetal direction and phloem elements centrifugally. The other segment produces only thin-walled parenchyma on both xylem and phloem side. In mature stems, some of the axial parenchyma embedded deep inside the xylem acquires meristematic activity and leads to the formation of thick-walled xylem derivatives centrifugally and phloem elements centripetally. The secondary xylem comprises vessel elements, tracheids, fibres and axial parenchyma. Rays are uni-multiseriate in the region of cambium that produces xylem and phloem derivatives, while in some of the regions of cambium large multiseriate, compound, aggregate and polycentric rays can be noticed.     

Water relations in silver birch during springtime: How is sap pressurised?

• Positive sap pressures are produced in the xylem of birch trees in boreal conditions during the time between the thawing of the soil and bud break. During this period, xylem embolisms accumulated during wintertime are refilled with water. The mechanism for xylem sap pressurization and its environmental drivers are not well known.
• We measured xylem sap flow, xylem sap pressure, xylem sap osmotic concentration, xylem and whole stem diameter changes, and stem and root non‐structural carbohydrate concentrations, along with meteorological conditions at two sites in Finland during and after the sap pressurisation period.
• The diurnal dynamics of xylem sap pressure and sap flow during the sap pressurisation period varied, but were more often opposite to the diurnal pattern after bud burst, i.e. sap pressure increased and sap flow rate mostly decreased when temperature increased. Net conversion of soluble sugars to starch in the stem and roots occurred during the sap pressurisation period. Xylem sap osmotic pressure was small in comparison to total sap pressure, and it did not follow changes in environmental conditions or tree water relations.
• Based on these findings, we suggest that xylem sap pressurisation and embolism refilling occur gradually over a few weeks through water transfer from parenchyma cells to xylem vessels during daytime, and then the parenchyma are refilled mostly during nighttime by water uptake from soil. Possible drivers for water transfer from parenchyma cells to vessels are discussed. Also the functioning of thermal dissipation probes in conditions of changing stem water content is discussed.

     

Developmental Changes in the Anatomy of the Sugarcane Stem in Relation to Phloem Unloading and Sucrose Storage

Transverse sections of immature and mature sugarcane internodes were investigated anatomically with white and fluorescence light microscopy. The pattern of lignification and suberization was tested histo-chemically. Lignification began in the xylem of vascular bundles and progressed through the sclerenchymatic bundle sheath into the storage parenchyma. Suberization began in parenchyma cells adjacent to vascular bundle sheaths and spread to the storage parenchyma and outer sheath cells. In mature internodes most of the storage parenchyma was lignified and suberized to a significant degree, except in portions of walls of isolated cells. The pattern of increasing lignification and suberization in maturing internodes more or less paralleled an increase of sucrose in stem tissue. In mature internodes having a high sucrose concentration, the vascular tissue was surrounded by thick-walled, lignified and suberized sclerenchyma cells. The apoplastic tracer dyes triso-dium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) and amido black 10 B, fed into cut ends of the stalk, wereconfined to the vascular bundles in all internodes above the one that was cut — with no dye apparently in storage parenchyma tissue. Thus both structural and experimental evidence is consistent with vascular tissue being increasingly isolated from the storage parenchyma as maturation of the tissue proceeds. We conclude that in mature internodes the pathway for sugars from the phloem to the storage parenchyma is symplastic. The data suggest that an increasingly greater role for a symplastic pathway of sugar transfer occurs as the tissue undergoes lignification/suberization.     

Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.     

Architectural and biochemical changes in embryonic tissues of maize under cadmium toxicity

Heavy metals greatly alter plant morphology and architecture, however detailed mechanisms of such changes are not fully explored. Two experiments were conducted to investigate the influence of cadmium (CdCl₂·2.5H₂O) on some germination, morphological, biochemical and histological characteristics of developing embryonic tissue of maize. In the first experiment, maize seeds were germinated in increasing levels of CdCl₂ (200–2000 μm ) in sand and measurements were taken of changes in germination and seedling development attributes. Based on these parameters, 1000 μM CdCl₂ was chosen for detailed biochemical and histological measurements. In the second experiment, seeds were germinated in Petri dishes and supplied with 0 (control) or 1000 μM CdCl₂ (Cd‐treated). Radicle, plumule, coleoptile and coleorhiza were measured for biochemical and histological changes. The highest amount of Cd was in the coleorhiza and radicle. Free proline, soluble sugars, anthocyanin, soluble phenolics, ascorbic acid, H₂O₂ and MDA were significantly higher in coleorhizae, followed by the coleoptile, radicle and plumule. Although the radicle and coleorhiza were relatively poor targets of Cd than the other tissues, Cd stress reduced cortical cell size and vascular tissues, and deformed xylem and phloem parenchyma in all plant parts. In conclusion, the main reason for reduced germination was the influence of Cd on architecture of the coleorhiza and coleoptile, which was the result of oxidative stress and other physiological changes taking place in these tissues.     

Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion

Sodium exclusion from leaves is an important mechanism for salt tolerance in durum wheat. To characterize possible control points for Na(+) exclusion, quantitative cryo-analytical scanning electron microscopy was used to determine cell-specific ion profiles across roots of two durum wheat genotypes with contrasting rates of Na(+) transport from root to shoot grown in 50 mm NaCl. The Na(+) concentration in Line 149 (low transport genotype) declined across the cortex, being highest in the epidermal and sub-epidermal cells (48 mm) and lowest in the inner cortical cells (22 mm). Na(+) was high in the pericycle (85 mm) and low in the xylem parenchyma (34 mm). The Na(+) profile in Tamaroi (high transport genotype) had a similar trend but with a high concentration (130 mm) in the xylem parenchyma. The K(+) profiles were generally inverse to those of Na(+). Chloride was only detected in the epidermis. These data suggest that the epidermal and cortical cells removed most of the Na(+) and Cl(-) from the transpiration stream before it reached the endodermis, and that the endodermis is not the control point for salt uptake by the plant. The pericycle as well as the xylem parenchyma may be important in the control of net Na(+) loading of the xylem.