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Michael Wisniewski

Frost-sensitive plant species have a limited ability to tolerate ice formation in their tissues. Most plants can supercool below 0°C and avoid ice formation. Discrepancies exist about the role of intrinsic and extrinsic ice-nucleating agents in initiating ice formation in plants. Previous research has demonstrated the ability of infrared video thermography to directly observe and record the freezing process in plants (Wisniewski et al., 1997. Plant Physiol. 113:4378–4397). In the present study, the ability of droplets of a suspension of the ice-nucleating-active (Ice+) bacterium, Pseudomonas syringae, and droplets of deionized water, to induce ice formation in bean plants was compared. The activity of these agents were also compared to intrinsic ice formation in dry plants. Results indicated that the presence of the Ice+ bacteria in droplets ranging from 0.5–4.0 μL always induced freezing at a warmer temperature than droplets of deionized water alone (no bacteria) or intrinsic nucleators in dry plants. When droplets of Ice+ bacteria were allowed to dry, they were no longer effective but were active again upon rewetting. Droplets of water would often supercool below temperatures at which ice formation was initiated by intrinsic agents. When a silicon grease barrier was placed between the droplets of Ice+ bacteria and the leaf surface, the bacteria were no longer capable of inducing ice formation in the plant, despite the droplets being frozen on the plant surface. This indicates that ice crystals must penetrate the cuticle in order to induce freezing of the plant.

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Rajeev Arora and Michael Wisniewski

The seasonal pattern of dehydrin accumulation was characterized during cold acclimation and deacclimation in the xylem tissues of genetically related (sibling) deciduous and evergreen peach (Prunus persica L.). Immunological studies indicate that a 60-kD polypeptide in peach xylem tissues is a dehydrin protein. Comparison of its accumulation pattern with seasonal fluctuations in cold hardiness indicate that dehydrin accumulated to high levels during the peak of cold acclimation. However, its accumulation was only weakly associated with cold hardiness during early stages of cold acclimation and during deacclimation. Our results indicate that factors related to supercooling rather than dehydrin accumulation may be primarily responsible for determining levels of cold hardiness during transition periods.

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Michael Wisniewski and Les Fuchigami

During autumnal leaf senescence, leaf nitrogen is translocated to bark and root tissues for storage. By definition, proteins that accumulate in large amounts in winter and are absent in summer are called storage proteins. These storage proteins are believed to play an important role in spring growth and helping trees to tolerate and/or recover from both abiotic and biotic stress. Little knowledge exists regarding storage proteins in apple, their physiological function, or how management practices impact them. Our objectives in this research was to characterize seasonally regulated proteins in apple, develop knowledge about their physiological function, and determine how they are affected by management practices. Results of the first-year studies have identified four major proteins that exhibit a seasonal pattern of accumulation in bark tissues of apple. One of these is a pathogenesis-related protein, meaning that it plays a role in disease resistance. Another of these proteins is a stress-related protein important in the use of carbohydrates under stress conditions. A third protein is a vegetative storage protein serving as a reserve for nitrogen. The last protein has not been completely identified. Greatest seasonal fluctuation of these proteins occurred in current season and 1-year-old bark tissues. Experimental studies that achieved varying levels of nitrogen in shoot tissues of young Fuji apple trees were examined for the effect on the accumulation of these proteins. Results indicated that despite a significant increase in total nitrogen, increases in the accumulation of these proteins were only slight. Instead, it appears that most of the nitrogen was present as free amino acids rather than stable proteins. These data indicate that more knowledge is required to determine the benefits and feasibility of elevating the levels of specific proteins in dormant apple trees or trying to manipulate the type of amino acids that accumulate.

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Timothy Artlip and Michael Wisniewski

Dehydrins are desiccation-induced proteins. Many plants have several dehydrin genes, some of which are primarily cold induced while others are primarily abscisic acid (ABA) or desiccation induced. Only one dehydrin gene (ppdhn1) has been reported in peach. The dehydrin gene is seasonally regulated and associated with cold acclimation. Because molecular markers for desiccation resistance may aid in the selection of drought- and cold-tolerant genotypes, we sought to determine if ppdhn1 was inducible by desiccation and ABA in all tissues (i.e., a whole-plant response) and to examine the relationship between expression of ppdhn1, desiccation, and dehydrin protein (PCA60). One-year-old `Rio Oso Gem' peach [Pranus persica (L.) Batsch.] trees were maintained at a stem water potential of -2.0 MPa by withholding water for 1 week, followed by daily watering for 1 week for some of the trees. ABA (100 mm) was applied to similar trees that were well watered. Total RNA and protein were extracted from bark, leaves, xylem, and roots, fractionated by electrophoresis, blotted to membranes, and probed with either a peach-specific dehydrin cDNA clone or polyclonal antibodies directed against dehydrin. Accumulation of ppdhn1/PCA60 was induced more by desiccation than ABA applications. Additionally, such accumulation was tissue dependent, being highest in bark tissues and lowest in leaf tissues. The presence of ppdhn1 transcript and corresponding PCA60 protein were not always commensurate with each other. In particular, elevated levels of PCA60 were still present 1 week after desiccation recovery when transcript levels had decreased significantly or were undetectable, indicating that dehydrin is a stable protein. In general, our data indicate that ppdhn1 is similar to other cold-induced dehydrins that are only slightly induced by ABA. In contrast to cold-induced dehydrins, ppdhn1 was strongly induced by desiccation. While synthesis of dehydrin is tightly associated with the onset of stress, disappearance and turnover seem less linked to alleviation of the inducing stress.

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Michael Wisniewski and Glen Davis

The pit membrane of xylem parenchyma of peach plays an important role in deep supercooling. Enzyme hydrolysis of xylem tissue indicated that the pit membrane is rich in pectin. The objective of the present study was to determine if removal of calcium from the cell wall would effect deep supercooling by loosening the cell wall. Current year shoots of `Loring' peach were infiltrated with oxalic acid, EGTA, or sodium phosphate buffer for 24-48 hours and then prepared for either ultrastructural analysis or differential thermal analysis. The use of 5-50 mM oxalic acid resulted in a distinct reduction in the size of the low-temperature exotherm (LTE) with increasing concentration. Oxalic acid also produced a loosening and swelling of the pit membrane. The use of EGTA (100 mM) or NaP04 (150 mM) produced only a slight shift in the LTE to warmer temperatures when compared to fresh tissues. Heat treatments (30-100°C) also resulted in a gradual shift of the LTE to warmer temperatures. The data indicate that cross-linking of pectins may play a role in defining the pore structure of the pit membrane and that this area of the cell wall plays an integral role in deep supercooling of peach wood.

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Leslie H. Fuchigami and Michael Wisniewski

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Michael Wisniewski, Michael Glenn and Mick Fuller

Most plants exhibit the ability to supercool to some extent without freezing. The extent of supercooling, however, is limited by the action of intrinsic and extrinsic ice nucleating agents which initiate ice formation and propagation within a plant at relatively warm subzero temperatures (-1.5 to -3.5 °C). In herbaceous plants, extrinsic ice-nucleating agents (such as ice-nucleation bacteria, dew, and other good nucleating agents) significantly limit the ability to supercool below 0 °C. It is believed that with an absence of these extrinsic nucleating agents that plants could supercool to less than -4 °C. Other evidence indicates that intrinsic nucleating agents may also significantly limit the extent of supercooling. Questions also exist about nucleation in woody plants and especially the new growth (flowers, leaves, and shoots) present in spring. A better understanding of how freezing is initiated in plants has been limited by the inability to determine and visualize the initial site of ice nucleation and pattern of ice propagation. We have used infrared video thermography to study freezing in young tomato (Lycopersicon esculentum) plants and to determine if a hydrophobic barrier on the plant surface could prevent the action of extrinsic nucleating agents such as Ice + bacterial strain (Cit7) of Pseudomonas syringae from initiating freezing within a plant. Tomato plants were grown in a greenhouse in individual pots and used when they were 4 to 6 weeks old. Freezing tests were conducted in a programmable freezing chamber, and freezing was visualized and recorded on videotape using an infrared radiometer. Freezing of the plants was extrinsically induced by the application of droplets (5 μl) of water containing Cit7. To provide a barrier to the action of extrinsic ice-nucleating agents, an emulsion of hydrophobic kaolin was applied to the plant surface before applying an extrinsic nucleating agent. Results indicate that dry, young tomato plants can supercool to as low as -6 °C whereas plants having a single droplet of Cit7 would freeze at -1.5 to -2.5 °C. Applying the hydrophobic barrier blocked the effect of Cit7 and allowed the plants to also supercool to -6 °C, despite the presence of frozen droplets. Experiments under natural freezing conditions are in progress.

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Michael Wisniewski, D. Michael Glenn, Lawrence Gusta and Michael P. Fuller

Factors that determine when and to what extent a plant will freeze are complex. Although thermocouples have served as the main method of monitoring the freezing process in plants, infrared (IR) thermography offers distinct advantages and the use of this latter technology has provided new insights on the processes of ice nucleation and propagation. This technology is based on the fact that freezing is an exothermic event. The temperature and spatial resolution of a high-resolution IR camera has enabled researchers to clearly define initial sites of nucleation as well as monitor the ice front as it spreads into surrounding tissues. Ice nucleation is induced by both extrinsic and intrinsic nucleators. Ice nucleation-active bacteria and moisture are two major extrinsic agents. In herbaceous plants, the influence of extrinsic ice nucleators on ice nucleation can be moderated by thick cuticles or the application of synthetic hydrophobic barriers. The situation in woody plants, however, is different. Woody plants appear to possess native, intrinsic nucleating agents that are as active as many extrinsic agents. However, the identity of the intrinsic nucleating agents in woody plants is not known. Despite the presence of intrinsic nucleating agents, barriers exist in woody plants that inhibit growth of ice from older stems into primary, lateral appendages. This is important because many tissues in woody plants that are frost-sensitive are flowers and primary, elongating shoot tissues that arise from buds attached to older stems. Pictures derived from video segments of the freezing process and data on the ability to block nucleation through the use of hydrophobic kaolin are provided.

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Michael Wisniewski, D. Michael Glenn and Michael P. Fuller

Extrinsic ice nucleating agents (such as ice-nucleation-active bacteria, dew, etc.) significantly limit the ability of herbaceous plants to supercool. It is believed that with an absence of these extrinsic nucleating agents, a plant could supercool to less than -4 °C. Other evidence, however, indicates that intrinsic nucleating agents may limit the extent of supercooling. Infrared video thermography was used to study freezing in young, `Rutgers' tomato (Lycopersicon esculentum L.) plants and to determine if a hydrophobic barrier on the plant surface could prevent extrinsic nucleating agents such as Ice+ bacterial strain (Cit7) of Pseudomonas syringae Van Hall from initiating freezing within a plant. Freezing tests were conducted in a programmable freezing chamber, a radiative frost chamber, and outdoors. Freezing was visualized and recorded on videotape using an infrared radiometer. Freezing of the plants was induced extrinsically by application of droplets (5 to 7 μL) of water containing Cit7. To provide a barrier to the action of extrinsic ice nucleating agents, an emulsion of hydrophobic kaolin (aluminum silicate mineral) was applied to the plant surface before application of an extrinsic nucleating agent. Results indicate that dry, young tomato plants can supercool to as low as -6 °C whereas plants having a single droplet of Cit7 would freeze at -1.5 to -2.5 °C. Application of the hydrophobic barrier blocked the effect of Cit7 and allowed whole plants to also supercool to -6 °C, despite the presence of frozen droplets on the leaf surface. When whole plants were sprayed with water and Cit7 using an aerosol sprayer and exposed to -3 °C, plants coated with the hydrophobic particle film exhibited significantly less foliar injury then nontreated plants. Similar results were obtained using the radiative frost chamber. Experiments conducted under natural frost conditions also resulted in less injury to the coated plants. The hydrophobic kaolin particle film performed better at preventing plants from freezing due to extrinsic ice nucleation than nonaltered, hydrophyllic kaolin alone or an antitranspirant with putative frost protection properties.