Deep Undercooling in Woody Taxa Growing North of the -40 degrees C Isotherm

The freezing of deep undercooled water in cold-hardened 3-year-old stems of 16 woody taxa was studied in mid-January by differential thermal analysis. The initiation temperature and the size of the low temperature exotherm (LTE) were compared for nonthawed, thawed, and freeze-killed stems. In general, the initiation temperature of the LTE for nonthawed stems occurred at a lower temperature than for thawed stems and freeze-killed stems. In some cases, no LTE was detected in nonthawed stems although a LTE was detected after thawing. The size of the LTE increased after thawing the stem and also after the stem was freeze killed. The LTE observed in one species disappeared upon exposure to continuous low sub-zero temperatures. Results suggest that undercooling which subsequently results in the LTE in woody stems is due to the cell wall and the plasma membrane. During periods of prolonged freezing, cellular water migrates from the cells which undercool to extracellular ice. This results in a concentration of cell solutes which lowers the homogeneous nucleation temperature of the cell sap. The cold hardiness of nonthawed and thawed stems was compared by a controlled freeze test. In general, thawing had little effect on the survival temperature whereas it had a marked effect on the initiation of the LTE.     

Nucleic Acid and protein changes in relation to cold acclimation and freezing injury of korean boxwood leaves

Quantitative and qualitative differences in nucleic acids of Korean boxwood (Buxus microphylla var. Koreana) leaves were determined by methylated albumin kieselguhr chromatography at different levels of cold hardiness. During cold acclimation there was an increase in RNA, mainly ribosomal RNA, with little or no change in DNA. The increase in ribosomal RNA was closely paralleled by an increase in water soluble and membrane bound proteins. As cold hardiness increased, ribonuclease activity declined.     

Molecular modeling of the (S)-ABA binding site of the receptor involved in the induction of freezing tolerance: A hypothetical receptor model

Molecular modeling was used to compare abscisic acid (ABA) analogs to rationalize reported structure-activity relationships based on the induction of freezing tolerance in bromegrass (Bromus inermis Leyss.) cell culture. A modified version of the active analog approach was employed with (S)-ABA used as the standard to which other compounds were superimposed. Common conformations that present similar three-dimensional steric and electronic patterns were identified through conformational searches. From this analysis, a hypothetical model for the putative (S)-ABA receptor was constructed with the following features. The ring is in the pseudochair conformation with an axial side chain. The C-1 has the same absolute stereochemistry as (S)-ABA. The model suggests that discrimination between (S)-ABA, phaseic acid, and (R)-ABA is due to the presence of an ether bridge or a methyl group below the C-2 of (S)-ABA, in a region proposed to be occupied by the receptor. The side chain is syn with the ring and positioned approximately above the 2-carbon of the ring. The model serves as a working hypothesis for testing receptor requirements and can be used to direct future analog studies.The following convention will be used throughout this paper: the ring face from which the side chain extends will be referred to as the upper face. This will simplify later discussion of analogs with multiple stereocenters that interact with a common active site. From this it follows that the upper face of (S)-ABA is the -face, and in (R)-ABA it is the -face (nomenclature of Rose et al., 1980).     

Structure-Activity Relationships of Abscisic Acid Analogs Based on the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Cell Cultures

The induction of freezing tolerance in bromegrass (Bromus inermis Leyss) cell culture was used to investigate the activity of absisic acid (ABA) analogs. Analogs were either part of an array of 32 derived from systematic alterations to four regions of the ABA molecule or related, pure optical isomers. Alterations were made to the functional group at C-1 (acid replaced with methyl ester, aldehyde, or alcohol), the configuration at C-2, C-3 (cis double bond replaced with trans double bond), the bond order at C-4, C-5 (trans double bond replaced with a triple bond), and ring saturation (C-2′, C-3′ double bond replaced with a single bond so that the C-2′ methyl and side chain were cis). All deviations in structure from ABA reduced activity. A cis C-2, C-3 double bond was the only substituent absolutely required for activity. Overall, acids and esters were more active than aldehydes and alcohols, cyclohexenones were more active than cyclohexanones, and dienoic and acetylenic analogs were equally active. The activity associated with any one substituent was, however, markedly influenced by the presence of other substituents. cis, trans analogs were more active than their corresponding acetylenic analogs unless the C-1 was an ester. Cyclohexenones were more active than cyclohexanones regardless of oxidation level at C-1. An acetylenic side chain decreased the activity of cyclohexenones but increased the activity of cyclohexanones relative to their cis, trans counterparts. Trends suggested that for activity the configuration at C-1′ has to be the same as in (S)-ABA, in dihydro analogs the C-2′-methyl and the side chain must be cis, small positional changes of the 7′-methyl are tolerable, and the C-1 has to be at the acid oxidation level.     

Plant Cold Acclimation: The Role of Abscisic Acid

The freezing tolerance or cold acclimation of plants is enhanced over a period of time by temperatures below 10°C and by a short photoperiod in certain species of trees and grasses. During this process, freezing tolerance increases 2–8°C in spring annuals, 10–30°C in winter annuals, and 20–200°C in tree species. Gene upregulation and downregulation have been demonstrated to be involved in response to environmental cues such as low temperature. Evidence suggests ABA can substitute for the low temperature stimulus, provided there is also an adequate supply of sugars. Evidence also suggests there may be ABA-dependent and ABA-independent pathways involved in the acclimation process. This review summarizes the role of ABA in cold acclimation from both a historical and recent perspective. It is concluded that it is highly unlikely that ABA regulates all the genes associated with cold acclimation; however, it definitely regulates many of the genes associated with an increase in freezing tolerance.     

Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants

A simplified method for the isolation of a plasma membrane-enriched fraction from plants utilizing an aqueous two-polymer phase system is outlined. Mainly, the plant used was Orchard grass (Dactylis glomerata L.). The two-phase system consisted of 5.6% (w/w) of dextran T500 and 5.6% (w/w) of polyethyleneglycol 4000 in 0.5 molar sorbitol-15 millimolar Tris-maleate (pH 7.3), and 30 millimolar NaCl. In this system, the plasma membranes and the other membranes were preferentially partitioned into the top phase and into the lower phase, respectively. The purity of the isolated plasma membrane was sufficiently high even after a single partition (i.e. about 85% purity) and more than 90% purity was obtained after repeating the partition in a newly prepared lower phase. The plasma membrane was identified with the aid of phosphotungstic acid-chromic acid stain and the association of vanadate-sensitive Mg²⁺-ATPase. The plasma membrane-associated ATPase had a pH optimum at 6.5 and showed a high specificity for Mg²⁺ and ATP. KCl stimulation was low (6% stimulation) at the pH optimum, but a relatively high stimulation (23%) occurred at pH 5.5. This method for plasma membrane isolation may be applicable to a wide variety of plants and plant tissue including green leaves.     

Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses

BACKGROUND AND AIMS: Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. METHODS: Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. KEY RESULTS: Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. CONCLUSIONS: The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Monoubiquitinated histone H1B is required for antiviral protection in CD4(+)T cells resistant to HIV-1

Linker histone H1B (H1B) coeluted with an antiviral activity during the purification of HIV-1 resistance factor (HRF) from supernatants of HRF(+) cells. Western blot analysis of the supernatant using alpha-H1 and alpha-ubiquitin antibodies detected the same band of roughly 46 kDa; this band was absent from the control supernatant. Depletion of histone from biologically active material did not affect its potential, suggesting that ubiquitinated H1B is not required for the HRF-mediated antiviral protection in HIV-1 susceptible target cells; however, specific silencing of histone H1B via RNAi in HRF(+) cells reduced the biological activity of cell culture supernatants by 96% and reversed the HIV-1 resistance phenotype of HRF(+) cells. Exposure to HRF induced ubiquitination and secretion of H1B from target HIV-1 susceptible cells, suggesting that ubiquitinated H1B is a cofactor of HRF, possibly regulating its expression and secretion from CD4(+)T cells induced to resist HIV-1 infection.