Xylem Ray Parenchyma Research Articles

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.

Ultrastructural study of deep supercooling of xylem ray parenchyma cells from Styrax obassia

Some of the water in the xylem of a woody species ( Styrax obassia Sieb. et Zucc.), collected during the summer, appeared to exhibit deep supercooling and the breakdown of supercooling at around - 20°C, as determined by differential thermal analysis (DTA). Ultrastructural observations of the low-temperature behaviour of the xylem, using both cryo-scanning electron microscopy (cryo-SEM) and freeze-fracture replica techniques, provided direct evidence that xylem ray parenchyma cells can be deeply supercooled to around - 20°C and that the breakdown of supercooling produces intracellular freezing in the ray parenchyma cells. Ultrastructural observations also confirmed that the supercooling of ray parenchyma cells was basically unaffected by the presence or absence of bulk water in the neighbouring tracheary elements, and that individual ray parenchyma cells acted as isolated droplets of water. Furthermore, the present ultrastructural study provides clear evidence that intracellular freezing upon the breakdown of deep supercooling results in severe damage to plasma membranes. Nontheless, the characteristics of supercooling reappeared in the ray parenchyma cells upon recooling.

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.

Response of Woody Plant Cells to Dehydrative Stress

Low temperature scanning electron microscopy was used to evaluate changes in cell and tissue morphology in response to a dehydrative stress. Bark cortical cells of both Cornus florida L. (flowering dogwood) and Cornus sericea L. (red osier dogwood) were collapsed at low tissue moisture. Bark cortical cell volume was reduced, and cell walls appeared buckled and distorted. In contrast, the shape and volume of xylem ray parenchyma cells did not appear to change in response to the dehydrative stress treatment. There was no evidence of cell wall buckling, distortion, or separation in either of the two species. Instead the xylem ray parenchyma cells within stressed tissue appeared the same as controls. These observations were similar to those reported regarding the response of woody tissues during freezing. The results do not support the hypothesis that rigid cell walls are a characteristic of plants that exhibit deep supercooling and that differences in cell wall rigidity distinguish species that supercool from species that do not.

CRYOPRESERVATION OF DORMANT BUDS FROM COLD-TENDER TAXA USING A MODIFIED VITRIFICATION PROCEDURE

Cryopreservation of woody-plant, dormant buds may provide cost-effective, long-term, back-up conservation of germplasm for vegetatively propagated crops that are presently maintained as trees in field gene banks. Dormant buds can be recovered quickly by grafting to dwarfing rootstocks, thus producing flowers for breeding purposes, with minimum potential for inducing somaclonal variants. These attributes are essential to preserve the clonal integrity of unique gene combinations such as those found in tree fruit crops. Previous research has shown that dormant buds from cold-hardy apples can be recovered from storage in liquid nitrogen (LN) with high survival rates (80% to 100%) using controlled desiccation and slow freezing before immersing in LN. On the other hand, dormant buds from cold-tender taxa and buds collected at less than optimal stages for desiccation and freezing have much lower (0% to 50%) survival rates. We increased survival of cold-tender taxa by using a modified vitrification procedure. Dormant apple buds from tender and hardy cultivars were perfused with modified PVS [15% (w/v) ethylene glycol, 15% (w/v) propylene glycol, 7% (w/v) DMSO, and 15% (w/v) glycerol in 0.5 m sorbitol]. Toxicity from the PVS was reduced by dilution soaking in 1 m sorbitol, 0.2 m raffinose, and 15 mm CaCl2 before and after quench-freezing and slow-freezing cryopreservation. Up to 100% of some cold-tender taxa survived. In addition, xylem ray parenchyma tissues that supercool and are normally killed at about -40C with the desiccation protocol survived this vitrification procedure.

CHARACTERIZATION OF LOW TEMPERATURE DAMAGE AND SUBSEQUENT REGROWTH RESPONSE OF FIELD GROWN ROSES

Plants of field grown rose cultivars Blaze, Gold Glow, Queen Elizabeth, Mr. Lincoln, Montezuma, Don Juan, Chicago Peace, and Pink Peace endured two major freezes. Temperatures fell to -13°C on 16 December 1989 and as low as -20°C during an extended period from 17 to 28 December 1989 when the highest temperature reached was 5°. Grade 1 plants of each cultivar were harvested on 5 January 1990. At harvest, discoloration of the pith, xylem ray parenchyma and bud union tissue was assessed. Additional plants were then potted and forced in a glasshouse at 15° night temperalure with venting at 21° during the day. At the end of the initial flush of growth, which was defined as either the opening of the first flower or the determination that all new shoots were blind, new growth was rated and measured. Blaze exhibited minimal damage with only slight pith discoloration. The total number of flowering shoots (TNFS) for Blaze was 5.5 per plant which is an expected number from a grade 1 plant. Of the other cultivars. Gold Glow and Pink Peace exhibited pith, xylem, and bud union damage with up to 50% cane dieback, but produced flowering shoots from the graft union. However, only half the expected TNFS per plant were produced. The remaining cultivars also exhibited higher damage levels than Blaze which resulted in reduced shoot numbers and flowering. Only Blaze plants received an acceptable plant marketability rating.

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.

Plastids and glycolipids in the stemwood of Pinus sylvestris L.

The ultrastructure of plastids in xylem ray parenchyma cells of Pinus sylvestris L. was studied and compared with the glycolipid composition of the stemwood. Seasonal changes of the ultrastructure were studied by taking samples regularly throughout the year. The plastids resemble amyloplasts. They usually have one large starch grain, and considerable variation in structure and starch content was observed, especially in the innermost sapwood and in the sapwood-heartwood transition zone. Electron-dense deposits were observed attached to the plastid membranes and envelopes, especially in the transition zone, from April to November. The plastids were aggregated near the nucleus and the starch disappeared during the winter (January–March). The glycolipids, monogalactosyldiacylglycerol (MGDG) and di-galactosyldiacylglycerol (DGDG), were present only in the sapwood, in trace amounts. The glycolipid content was slightly greater in the outer sapwood than in the sapwood-heartwood transition zone. DGDG was the dominant lipid of the two.

Freezing avoidance by deep supercooling in hydrated lettuce seeds

DRY seeds of several species survive exposure to extremely low temperatures, including immersion in liquid air for up to 60 d (refs 1–3), but most young seedlings are very susceptible4. Fully hardened twigs of several northern tree species tolerate freezing in liquid nitrogen (−196 °C) (ref. 5), and deep supercooling seems to be an important mechanism for the avoidance of freezing in both xylem ray parenchyma and flower buds of many other hardy but less cold resistant, deciduous, woody species6,7. Resistance to freezing stress in seeds has been suggested to be related to water content1,4,8, but little attention has been paid to either the relationship between stage of germination and degree of freezing resistance or the nature of the resistance. We report here that hydrated lettuce seeds (achenes) can avoid injury by deep supercooling and that the level of resistance can be determined precisely by differential thermal analysis (DTA). Furthermore, we show that the deep supercooling is dependent on the intact structure of the endosperm.

Cold Hardiness and Deep Supercooling in Xylem of Shagbark Hickory

Differential thermal analysis, differential scanning calorimetry, pulsed nuclear magnetic resonance spectroscopy, and low temperature microscopy are utilized to investigate low temperature freezing points or exotherms which occur near -40 C in the xylem of cold-acclimated shagbark hickory (Carya ovata L.). Experiments using these methods demonstrate that the low temperature exotherm results from the freezing of cellular water in a manner predicted for supercooled dilute aqueous solutions. Heat release on freezing, nuclear magnetic resonance relaxation times, and freezing and thawing curves for hickory twigs all point to a supercooled fraction in the xylem at subfreezing temperatures. Calorimetric and low temperature microscopic analyses indicate that freezing occurs intracellularly in the xylem ray parenchyma. The supercooled fraction is found to be extremely stable, even at temperatures only slightly above the homogeneous nucleation temperature for water (-38 C). Xylem water is also observed to be resistant to dehydration when exposed to 80% relative humidity at 20 C. D(2)O exchange experiments find that only a weak kinetic barrier to water transport exists in the xylem rays of shagbark hickory.

The Relationship of Exotherms to Cold Injury in Apple Stem Tissues1,2

Abstract During controlled freezing as many as 4 exotherms were detected in apple twigs by differential thermal analysis (DTA). Three of the exotherms usually occurred at relatively high temperatures above the killing point of stem tissues. There was no apparent association between any of these exotherms and freezing injury to cambium, cortex, phloem, or vegetative buds. The temp of the fourth exotherm (D), however, coincided with the killing temp of xylem ray and pith parenchyma as hardiness changed during the season. DTA studies and hardiness tests performed on whole and dissected twigs indicated that the fourth exotherm arose from the xylem and pith tissues; that injury to xylem and pith occurred when twigs were frozen below the initiation temp of the fourth exotherm independent of cooling rate and that bud and bark tissues were considerably hardier than xylem in mid-winter while in early autumn and late spring xylem and pith were hardier than buds and bark. The results suggest that freezing injury to xylem and pith cells is caused by a specific freezing event, and that it is different from freezing injury to other stem tissues. Measurements of the fourth exotherm may provide a convenient way of studying freezing injury and resistance in xylem – a recurring problem in many fruit species.