Seizo Fujikawa

Cryo-scanning electron microscopic study on freezing behavior of xylem ray parenchyma cells in hardwood species

Differential thermal analysis (DTA) has indicated that xylem ray parenchyma cells (XRPCs) of hardwood species adapt to freezing of apoplastic water either by deep supercooling or by extracellular freezing, depending upon the species. DTA studies indicated that moderately cold hardy hardwood species exhibiting deep supercooling in the XRPCs were limited in latitudinal distribution within the -40 degrees C isotherm, while very hardy hardwood species exhibiting extracellular freezing could distribute in colder areas beyond the -40 degrees C isotherm. Predictions based on the results of DTA, however, indicate that XRPCs exhibiting extracellular freezing may appear not only in very hardy woody species native to cold areas beyond the -40 degrees C isotherm but also in less hardy hardwood species native to tropical and subtropical zones as well as in a small number of moderately hardy hardwood species native to warm temperate zones. Cryo-scanning electron microscopic (cryo-SEM) studies on the freezing behavior of XRPCs have revealed some errors in DTA. These errors are originated mainly due to the overlap between exotherms produced by freezing of water in apoplastic spaces (high temperature exotherms, HTEs) and exotherms produced by freezing of intracellular water of XRPCs by breakdown of deep supercooling (low temperature exotherms, LTEs), as well as to the shortage of LTEs produced by intracellular freezing of XRPCs. In addition, DTA results are significantly affected by cooling rates employed. Further, cryo-SEM observations, which revealed the true freezing behavior of XRPCs, changed the previous knowledge of freezing behavior of XRPCs that had been obtained by freeze-substitution and transmission electron microscopic studies. Cryo-SEM results, in association with results obtained from DTA that were reconfirmed or changed by observation using a cryo-SEM, revealed a clear tendency of the freezing behavior of XRPCs in hardwood species to change with changes in the temperature in the growing conditions, including both latitudinal and seasonal temperature changes. In latitudinal temperature changes, XRPCs in less hardy hardwood species native to tropical and subtropical zones exhibited deep supercooling to -10 degrees C, XRPCs in moderately hardy hardwood species native to temperate zones exhibited a gradual increase in the supercooling ability to -40 degrees C from warm toward cool temperate zones, and XRPCs in very hardy hardwood species native to boreal forests exhibited extracellular freezing via an intermediate form of freezing behavior between deep supercooling and extracellular freezing. In seasonal temperature changes, XRPCs in hardwood species native to temperate zones changed their supercooling ability from a relatively low degree in summer to a high degree in winter. XRPCs in hardwood species native to boreal forests changed their freezing behavior from deep supercooling to -10 degrees C in summer to extracellular freezing in winter. These results indicate that the freezing behavior of XRPCs in hardwood species tends to shift gradually from supercooling of -10 degrees C, to a gradual increase in the deep supercooling ability to -40 degrees C or less, and finally to extracellular freezing as a result of cold acclimation in response to both latitudinal and seasonal temperature changes. It is thought that these temperature-dependent changes in the freezing behavior of XRPCs in hardwood species are mainly controlled by changes in cell wall properties, although no distinct changes were detected by electron microscopic observations in cell wall organization between hardwood species or between seasons. Evidence of temperature-dependent changes in the freezing behavior of XRPCs in hardwood species provided by the results of studies using a cryo-SEM has indicated the need for further investigation to clarify cold acclimation-induced cell wall changes at the sub-electron microscopic level in order to understand the mechanisms of freezing adaptation.

Formation of multiplex lamellae by equilibrium slow freezing of cortical parenchyma cells of mulberry and its possible relationship to freezing tolerance

SummaryCortical parenchyma cells of mulberry (Morus bombycis Koidz. cv. Goroji) become extremely cold hardy in winter and can tolerate equilibrium freezing below −30 °C and subsequent immersion into liquid nitrogen. We show in this ultrastructural study that, in these extremely cold hardy cortical parenchyma cells of mulberry collected in winter, initiation of freezing at −5 °C resulted in the formation of multiplex lamellae (MPL) that completely covered the area beneath the plasma membrane. The MPL were produced by fusion of pre-existing vesicular endoplasmic reticulum (ER), via a reticular ER network. The completed MPL were composed of a parallel array of sheet-like ER cisternae. This structural reorganization of the ER was completed within 10 min upon freezing at −5 °C and was quickly reversed upon thawing. The same structural reorganization of the ER was produced by osmotic dehydration of the cortical tissues with a 2.7 osmol sorbitol solution at 20 °C. Thus, the structural reorganization of the ER upon freezing was, in fact, produced by dehydration. In winter samples, the formation of MPL with the initiation of freezing completely inhibited close apposition of membranes upon deep freezing that has been reported to be a cause of freezing injury via the production of ultrastructural changes in the plasma membrane. Similar but more or less incomplete MPL were produced by freezing or osmotic dehydration in cortical parenchyma cells collected in spring and autumn, and these MPL partly inhibited close apposition of membranes. MPL were not produced in the cells of mulberry collected in summer and close apposition of membranes occurred upon deep freezing. We speculate that the formation of MPL with the initiation of freezing might play a specific role in inhibiting the close apposition of membranes due to the specific nature of the cisternal membranes and might, consequently, be responsible for the high freezing tolerance of winter cells.

Determination of the Role of Cold Acclimation-Induced Diverse Changes in Plant Cells from the Viewpoint of Avoidance of Freezing Injury

Cold acclimation is a complex adaptive mechanism by which plants survive freezing in winter. During cold acclimation, diverse intracellular and extracellular changes occur. Although most of these changes are related to the acquirement of freezing tolerance, the exact role of these changes in the attainment of freezing tolerance is still unclear. In this review, we suggest the possible role of some of these cold acclimation-induced changes in relation with increased freezing tolerance from the viewpoint of inhibition of freezing injury produced by close approach of membranes.

Seasonal changes in dehydration tolerance of xylem ray parenchyma cells of stylax obassia twigs that survive freezing temperatures by deep supercooling

Xylem ray parenchyma cells ofStylax obassia twigs undergo supercooling to −26 °C as the terminal temperature of the low temperature exotherm in summer and to −41 °C in winter upon freezing. During supercooling, no evidence of cell dehydration was recognized; cell walls were uncollapsed, no plasmolysis occurred, no ultrastructural changes in the plasma membranes or in cytoplasmic structures occurred, and high survival rates were maintained. Osmotic manipulation with hypertonic solutions of sorbitol did, however, cause dehydration of ray parenchyma cells, in which shrunken protoplasts and plasmolysis were observed. Freeze-fracture replicas showed that, in summer samples, osmotic dehydration produced aparticulate domains accompanied by fracture-jumps in the plasma membranes, which are known as a symptom of dehydration-induced injury, but no such effects were seen in winter samples. By contrast, in winter samples, osmotic dehydration produced conformational changes in the endoplasmic reticulum, which are known as a feature of dehydration tolerance, however, these changes were not seen in the summer samples. These results indicate that the ray parenchyma cells inS. obassia have the ability to tolerate dehydration in winter and lose this ability in summer. Such tolerance is similar to the dehydration tolerance in plant cells that adapt to freezing by extracellular freezing.

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

SummaryBy 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.

Akinete Formation in Tribonema bombycinum Derbes et Solier (Xanthophyceae) in Relation to Freezing Tolerance

Tribonema bombycinum (Xanthophyceae), was examined. T. bombycinum shifted from vegetative cells to akinetes with starving by a prolonged batch culture, by culture with a diluted medium, or by culture with a single nutrient-deficient medium. In addition, akinetes developed by desiccation, but cold treatment at 4 C did not facilitate akinete formation. During starving, the vegetative cells, which had a large central vacuole in the protoplasm and thin cell walls, finally changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation by starving, the freezing tolerance (LT50) increased gradually from −3 C in vegetative cells to far below −30 C in akinetes. When vegetative cells were subjected to equilibrium freezing, their size shrank greatly and aparticulate domains accompanied by fracture-jump lesions developed in the plasma membranes. Akinetes subjected to equilibrium freezing showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes. The morphological changes in the process of akinete formation and the responses to equilibrium freezing resembled those of cold-acclimated terrestrial plants.

Loading process of sugars into cabbage petiole and asparagus shoot apex cells by incubation with hypertonic sugar solutions

SummaryThe freezing tolerance of cabbage petioles and asparagus shoot apexes was increased by preincubation with 0.8 M sugar solutions. In cabbage petioles with an initial freezing tolerance of −3 °C (temperature for 50% cell survival), as determined by both electrolyte leakage and fluorescein diacetate vital staining, the freezing tolerance was increased to −13 °C by incubation with sorbitol solutions for 3 h. In meristematic cells of asparagus shoot apexes with an initial freezing tolerance of −7.5 °C, as determined by fluorescein diacetate vital staining, the freezing tolerance was increased to −30 °C by incubation with 0.8 M sugar solutions for 3 h, although other cells in the shoot apexes were killed by higher freezing temperatures. During incubation of both cabbage petioles and asparagus shoot apexes with sugar solutions, sugars were intracellularly taken up by osmotically induced fluid-phase endocytotic vesicles, as indicated by comovement of Lucifer Yellows carbohydrazide (LYCH) observed with a confocal laser scanning microscope. The amounts of intracellularly taken up sugars increased concomitantly with the formation of endocytotic vesicles depending on the time of incubation in parallel with a gradual increase of freezing tolerance. However, the endocytotic vesicles and their contents were retained not only after prolonged incubation after maximum freezing tolerance had been achieved but also after recovery of these tissue cells to isotonic conditions or after freeze-thawing. These results suggest that although sugars are intracellularly taken up by endocytotic vesicles, they might be sequestered within vesicles, casting doubt on their protective role to the plasma membranes as a main site of freezing injury. The pretreatment with 1 mMp-chloromercuribenzenesulfonic acid (PCMBS), an inhibitor of sugar transport, reduced the amounts of intracellular sugar uptake without affecting the formation of endocytotic vesicles, suggesting that sugars were, at least partly, taken up by sugar transporters. In the pretreatment with PCMBS, the freezing tolerance of incubated tissues with sugar solutions was significantly reduced, although addition of PCMBS per se did not affect survival. These results suggest that sugars taken up by sugar transporters, rather than sugars taken up by endocytotic vesicles, are mainly responsible for the increased freezing tolerance of cabbage petioles and asparagus shoot apexes. Furthermore, we aimed to study the occurrence of fluid-phase endocytosis with LYCH in an isotonic condition. Our results indicated that uptake of LYCH by fluid-phase endocytotic vesicles was not detected microscopically in isotonic condition, although LYCH was spectrofluorimetrically taken up in isotonic condition. Spectrofluorimetric uptake of LYCH was inhibited by addition of probenecid, an anion transport inhibitor. These results suggest that in cabbage petioles and asparagus shoot apexes, LYCH is taken up by anion transport but not by fluid-phase endocytosis in isotonic condition, and uptake of LYCH by fluid-phase endocytosis is restricted to occur only in hypertonic condition.

Isolation and Characterization of Endoplasmic Reticulum from Mulberry Cortical Parenchyma Cells

The endoplasmic reticulum (ER) is the largest organelle of the endomembrane system and it has many physiological functions, such as the synthesis of proteins and lipids, addition of oligosaccharide chains, transport of proteins and membrane materials, and the regulation of cytosolic calcium concentrations. Classical literature distinguishes three ER subcompartments: rough ER, smooth ER, and the nuclear envelope. However, it has been suggested that the ER has a multifunctional nature and that specialized subregions in addition to the three classical domains exist in plant ER (1, 2).