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     

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.     

梅花(Prunus mume Sieb et Zucc)远缘杂种及其亲本的差热分析与冻害关系的研究

用差热分析(Differential thermal analysis: DTA)研究了山桃(Prunus davidiana)、杏(P. armeniaca)、青岛“粉红梅”(P.mume cv.'Fenhong Mei,)、“小绿萼”(P.mume cv.'Small Green Calyx')及其种问杂种“小绿萼”梅×山桃、青岛“粉红梅”×杏和杏×青岛“粉红梅”的低温放热(Low temperature exotherm)与冻害关系,以及皮部和木质部的冰冻类型(Freezing pattern)。在差热分析中,观察到亲本和杂种的木质部都有二次放热现象。低温放热后,引起木质部和髓射线薄壁细胞死亡,原生质膜透性急剧增加。在杂种与亲本之间,存在着明显的差异。分离的皮部却只出现一次高温放热(High temperature exotherm)。高温放热是与冻害无关的。文末讨论了梅花及其杂种在北京越冬的主要障碍及有关栽培措施。     

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.     

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.     

Ice formation and tissue response in apple twigs

Abstract. The response of apple twig tissue to a freezing stress was examined using a combination of low temperature scanning electron microscopy and freeze substitution techniques. Bark and wood tissues responded differently. In the bark, large extracellular ice crystals were observed in the cortex. The adjacent cortical cells collapsed and a large reduction in cell volume was observed. The extent of cell collapse throughout the bark was not uniform. Cells in the periderm, phloem and cambium exhibited little change in cell volume compared to cortical cells. Large extracellular ice crystals were not observed in the xylem or pith tissues. The xylem ray parenchyma and pith cells did not collapse in response to a freezing stress, but retained their original shape. The pattern of ice formation and cell response was not observed to change with season or the level of cold acclimation. This study supported the concept that bark and xylem tissues exhibit contrasting freezing behaviour. The observations were consistent with the idea that water in bark freezes extracellularly while water in xylem ray parenchyma and pith cells may supercool to temperatures approaching –40 °C prior to freezing intracellularly.     

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     

Changes in carbohydrates, ABA and bark proteins during seasonal cold acclimation and deacclimation in Hydrangea species differing in cold hardiness

Cold injury is frequently seen in the commercially important shrub Hydrangea macrophylla but not in Hydrangea paniculata. Cold acclimation and deacclimation and associated physiological adaptations were investigated from late September 2006 to early May 2007 in stems of field-grown H. macrophylla ssp. macrophylla (Thunb.) Ser. cv. Blaumeise and H. paniculata Sieb. cv. Kyushu. Acclimation and deacclimation appeared approximately synchronized in the two species, but they differed significantly in levels of mid-winter cold hardiness, rates of acclimation and deacclimation and physiological traits conferring tolerance to freezing conditions. Accumulation patterns of sucrose and raffinose in stems paralleled fluctuations in cold hardiness in both species, but H. macrophylla additionally accumulated glucose and fructose during winter, indicating species-specific differences in carbohydrate metabolism. Protein profiles differed between H. macrophylla and H. paniculata, but distinct seasonal patterns associated with winter acclimation were observed in both species. In H. paniculata concurrent increases in xylem sap abscisic acid (ABA) concentrations ([ABA](xylem)) and freezing tolerance suggests an involvement of ABA in cold acclimation. In contrast, ABA from the root system was seemingly not involved in cold acclimation in H. macrophylla, suggesting that species-specific differences in cold hardiness may be related to differences in [ABA](xylem). In both species a significant increase in stem freezing tolerance appeared long after growth ceased, suggesting that cold acclimation is more regulated by temperature than by photoperiod.     

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.     

ULTRASTRUCTURE OF THE SUBGLANDULAR CELLS FROM THE FOLIAR NECTARIES OF COTTON IN RELATION TO THE DISTRIBUTION OF PLASMODESMATA AND THE SYMPLASTIC TRANSPORT OF NECTAR

Foliar nectaries on the midveins of 7-cm leaves from cotton (Gossypium hirsutum L., cv. Stoneville 213) were examined by light and electron microscopy. The nectaries consist of external multicellular papillae and internal subglandular tissue that extends from the bases of the papillae to the vascular tissue of the midveins. The subglandular tissue is composed of small parenchyma cells; it does not contain sieve elements or xylem vessels. The parenchyma cells are rich in mitochondria, and their walls contain numerous pit fields having a high concentration of plasmodesmata. The absence of vascular tissue and the significance of the pit fields in the subglandular tissue are discussed in relation to symplastic transport of nectar secretions.     

Immunogold localization of pectins and glycoproteins in tissues of peach with reference to deep supercooling

Living xylem tissues and floral buds of several species of woody plants survive exposure to freezing temperatures by deep supercooling. A barrier to water loss and the growth of ice crystals into cells is considered necessary for deep supercooling to occur. Pectins, as a constituent of the cell wall, have been implicated in the formation of this barrier. The present study examined the distribution of pectin in xylem and floral bud tissues of peach (Prunus persica). Two monoclonal antibodies (JIM5 and JIM7) that recognize homogalacturonic sequences with varying degrees of esterification were utilized in conjunction with immunogold electron microscopy. Results indicate that highly esterified epitopes of pectin, recognized by JIM7, were the predominant types of pectin in peach and were uniformly distributed throughout the pit membrane and primary cell walls of xylem and floral bud tissues. In contrast, un-esterified epitopes of pectin, recognized by JIM5, were confined to the outer surface of the pit membrane in xylem tissues. In floral buds, these epitopes were localized in middle lamellae, along the outer margin of the cell wall lining empty intercellular spaces, and within filled intercellular spaces. JIM5 labeling was more pronounced in December samples than in July/August samples. Additionally, epitopes of an arabinogalactan protein, recognized by JIM14, were confined to the amorphous layer of the pit membrane. The role of pectins in freezing response is discussed in the context of present theory and it is suggested that pectins may influence both water movement and intrusive growth of ice crystals at freezing temperatures.     

A xylem sap retrieval pathway in rice leaf blades: evidence of a role for endocytosis?

The structure and transport properties of pit membranes at the interface between the metaxylem and xylem parenchyma cells and the possible role of these pit membranes in solute transfer to the phloem were investigated. Electron microscopy revealed a fibrillar, almost tubular matrix within the pit membrane structure between the xylem vessels and xylem parenchyma of leaf blade bundles in rice (Oryza sativa). These pits are involved primarily with regulating water flux to the surrounding xylem parenchyma cells. Vascular parenchyma cells contain large mitochondrial populations, numerous dictyosomes, endomembrane complexes, and vesicles in close proximity to the pit membrane. Taken collectively, this suggests that endocytosis may occur at this interface. A weak solution of 5,6-carboxyfluorescein diacetate (5,6-CFDA) was applied to cut ends of leaves and, after a minimum of 30 min, the distribution of the fluorescent cleavage product, 5,6-carboxyfluorescein (5,6-CF), was observed using confocal microscopy. Cleavage of 5,6-CFDA occurred within the xylem parenchyma cells, and the non-polar 5,6-CF was then symplasmically transported to other parenchyma elements and ultimately, via numerous pore plasmodesmata, to adjacent thick-walled sieve tubes. Application of Lucifer Yellow, and, separately, Texas Red-labelled dextran (10 kDa) to the transpiration stream, confirmed that these membrane-impermeant probes could only have been offloaded from the xylem via the xylem vessel-xylem parenchyma pit membranes, suggesting endocytotic transmembrane transfer of these membrane-impermeant fluorophores. Accumulation within the thick-walled sieve tubes, but not in thin-walled sieve tubes, confirms the presence of a symplasmic phloem loading pathway, via pore plasmodesmata between xylem parenchyma and thick-walled sieve tubes, but not thin-walled sieve tubes.     

Response of Xylem Ray Parenchyma Cells of Red Osier Dogwood (Cornus sericea L.) to Freezing Stress (Microscopic Evidence of Protoplasm Contraction)

Freezing behavior of wood tissue of red osier dogwood (Cornus sericea L.) cannot be explained by current concepts of freezing resistance. Previous studies indicated that water in wood tissue presumably froze extracellularly. However, it was observed that xylem ray parenchyma cells within these tissues could survive temperatures as low as -80[deg]C and the walls of these cells did not collapse during freezing (S.R. Malone and E.N. Ashworth [1991] Plant Physiol 95: 871-881). This observation was unexpected and is inconsistent with the current hypothesis of cell response during freezing. Hence, the objective of our study was to further examine the mechanism of freezing resistance of wood tissue of red osier dogwood. We studied freezing stress response of xylem ray parenchyma cells of red osier dogwood using freeze substitution and transmission electron microscopy. Wood samples were collected in winter, spring, and summer of 1992. Specimens were cooled from 0[deg]C to -60[deg]C at 5[deg]C/h. Freezing stress did not affect the structural organization of wood tissue. However, the xylem ray parenchyma cells showed two unique responses to a freezing stress: protoplasm contraction and protoplasm fragmentation. Protoplasm contraction was evident at all freezing temperatures and in tissues collected at different times of the year. Cells with fragmented protoplasm, however, were noticed only in tissues collected in spring and summer. Protoplasm contraction in winter tissue occurred without apparent damage to the protoplasm. In contrast, protoplasm contraction in spring and summer tissues was accompanied by substantial damage. No evidence of intracellular ice formation was observed in parenchyma cells exposed to freezing stress. Differences in protoplasm contraction and appearance of cells with fragmented protoplasm likely indicated seasonal changes in cold hardiness of the wood tissue of red osier dogwood. We speculate that the appearance of fragmented protoplasm may indicate that cells are being injured by an alternative mechanism in spring and summer.     

Possible Mechanisms Limiting Movement of Ralstonia solanacearum in Resistant Tomato Tissues

The distribution and appearance of Ralstonia solanacearum in the upper hypocotyl tissues of root‐inoculated tomato seedlings of resistant rootstock cultivar LS‐89 (a selection from Hawaii 7998) and susceptible cultivar Ponderosa were compared to clarify the mechanism that limits the movement of the bacterial pathogen in resistant tomato tissues. In stems of wilted Ponderosa plants, bacteria colonized both the primary and the secondary xylem tissues. Bacteria were abundant in vessels, of which the pit membranes were often degenerated. All parenchyma cells adjacent to vessels with bacteria were necrotic and some of them were colonized with bacteria. In stems of LS‐89 plants showing no discernible wilting symptoms, bacteria were observed in the primary xylem tissues but not in the secondary xylem tissues. Necrosis of parenchyma cells adjacent to vessels with bacteria was observed occasionally. The pit membranes were often thicker with high electron density. The inner electron‐dense layer of cell wall of parenchyma cells and vessels was thicker and more conspicuous in xylem tissues of infected LS‐89 than in xylem of infected Ponderosa or mock‐inoculated plants. Electron‐dense materials accumulated in or around pit cavities in parenchyma cells next to vessels with bacteria, and in vessels with bacteria. Many bacterial cells appeared normal in vessels, except for those in contact with the pit membranes. These results indicate that R. solanacearum moves from vessel to vessel in infected tissues through degenerated pit membranes and that restricted movement in xylem tissues was the characteristic feature in LS‐89. The limitation in bacterial movement may be related to the thickening of the pit membranes and/or the accumulations of electron‐dense materials in vessels and parenchyma cells.     

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.     

Plasmodesmata and pit development in secondary xylem elements

Developing pit membranes of secondary xylem elements in Drimys winteri, Fagus sylvatica, Quercus robur, Sorbus aucuparia, Tilia vulgaris and Trochodendron aralioides have been examined by transmission electron microscopy. Absence of plasmodesmata from the membranes of vessel elements and tracheids indicates that their pits develop independently of these structures. On the other hand, plasmodesmata are abundant in pit membranes between fibres, parenchyma cells, and combinations of these cell types in Fagus, Quercus and Tilia. In each case the plasmodesmata pass right through the developing pit membrane. In the case of Sorbus fibres, however, plasmodesmata were absent from the majority of pit membrane profiles seen in sections. Occasionally they were observed in large numbers associated with a swollen region on one side of the pit membrane between fibres and between fibres and parenchyma, radiating from a small area of the middle lamella. In the case of fibre to parenchyma pitting, this swelling was always found on the fibre side of the membrane, while on the other side a small number of plasmodesmata were present completing communication with the parenchyma cytoplasm. These observations are discussed with regard to the role of plasmodesmata in pit formation, and in the differentiation of the various cell types in secondary xylem. The significance their distribution may have for our understanding of xylem evolution is also discussed.     

Xylem function of arid-land shrubs from California, USA: an ecological and evolutionary analysis

Xylem traits were examined among 22 arid-land shrub species, including measures of vessel dimensions and pit area. These structural measures were compared with the xylem functional traits of transport efficiency and safety from cavitation. The influence of evolution on trait relationships was examined using phylogenetic independent contrasts (PICs). A trade-off between xylem safety and efficiency was supported by a negative correlation between vessel dimensions and cavitation resistance. Pit area was correlated with cavitation resistance when cross species data were examined, but PICs suggest that these traits have evolved independently of one another. Differences in cavitation resistance that are not explained by pit area may be related to differences in pit membrane properties or the prevalence of tracheids, the latter of which may alter pit area through the addition of vessel-to-tracheid pits or through changes in xylem conduit connectivity. Some trait relationships were robust regardless of species ecology or evolutionary history. These trait relationships are likely to be the most valuable in predictive models that seek to examine anatomical and functional trait relationships among extant and fossil woods and include the relationship among hydraulic conductivity and vessel diameter, between vessel diameter and vessel length, and between hydraulic conductivity and wood density.     

Comparison of structural damage caused by Russian wheat aphid (Diuraphis noxia) and Bird cherry-oat aphid (Rhopalosiphum padi) in a susceptible barley cultivar, Hordeum vulgare cv. Clipper

The Russian wheat aphid (RWA, ( Diuraphis noxia ) and the Bird cherry-oat aphid (BCA, ( Rhopalosiphum padi L.) cause severe damage to grain crops, including barley. An investigation of the effects of these aphids on a susceptible cultivar revealed that BCA-infested barley plants remained healthy looking for 2 weeks after feeding commenced. In contrast, signs of stress and damage, including chlorosis and leaf necrosis were evident in RWA-infested plants. Our study suggests that damage to the vascular tissue because of sustained feeding by BCA was not as extensive as that caused by RWA. In addition, there is a marked difference in the salivary secretion pattern within xylem elements punctured by aphids tapping the xylem for water. RWA deposit electron-dense, amorphous to smooth saliva, which completely encases the inner walls of affected elements, and saliva encases pit membranes between xylem elements, and between xylem vessels and xylem parenchyma. Xylem tapped by BCA contained more granular saliva, which apparently does not occlude vessel wall apertures or the pit membranes to the same extent, as was observed with RWA. Damage to phloem tissue, including phloem parenchyma elements, sieve tube–companion cell (CC–ST) complexes as well as thick-walled ST, was extensive. Plasmodesmata between phloem parenchyma elements as well as pore plasmodesmata between the CC and ST were occluded by callose. We conclude that severe, perhaps permanent damage to conducting elements in RWA-infested leaves may be responsible for the detrimental chlorosis and necrosis symptoms. These symptoms are absent in BCA-infested plants.     

Regeneration of tissue following cavity formation in the vascular cylinders of Pisum sativum (Fabaceae) primary roots

The reorganization of vascular cylinders of pea (Pisum sativum, cv. Alaska) primary roots following the formation of vascular cavities was examined by light and electron microscopy. Cavities usually began forming ~20 mm from the root tip and were continuous to ~90 mm from the tips in roots 150 mm long, where they began filling with specialized parenchyma cells (SP cells). SP cells were usually produced by enlargement of parenchymous cells of the primary xylem at cavity margins. Depending on the extent and shape of the cavity, they were also sometimes produced by primary phloem parenchyma and early derivatives of the vascular cambium. Enlargement and some divisions of SP cells continued until a cavity was completely filled by them. SP cells proceeded through a series of cytoplasmic changes as they developed. First the cytoplasmic layer became thicker and more electron dense than surrounding cells. As SP cells enlarged there was an increase in vesicular traffic and the cytoplasm became less electron dense. Ultimately the cytoplasm thinned further, organelles degenerated, and the tonoplast sometimes broke down. SP cells did not form secondary walls. X-ray microanalysis revealed that SP cells accumulated potassium and rubidium to the same degree as cortical and xylem parenchyma cells and to a greater degree than immature secondary and late-maturing tracheary elements.     

Freezing in Conifer Xylem: II. PIT ASPIRATION AND BUBBLE FORMATION

A scanning electron microscope equipped with a freezing stagewas used to examine the effects of slow freezing on pit aspirationand bubble formation in living tree stems. The size (approximately 2.0 µm diameter) and the sphericalor ellipsoidal shape of the bubbles found in the centre of frozenlumens indicated freezing rates greater than 25 µm s^–1.Both unaspirated and aspirated bordered pits were found in thefrozen xylem. The technique used did not reveal enough pitsto determine whether unaspirated pits were more prevalent thanaspirated pits. These results are compared with hypotheses and results fromprevious work on freezing in conifer xylem. Key words: Freezing, conifer xylem, bordered pits, bubbles