Edward N. Ashworth

Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affecting both EW and cuticle deposition. The cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (WT-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor. The reduction in EW and cuticle deposition increased susceptibility to the fungal pathogen Exserohilum turcicum. Evidence suggests that this recessive mutation occurs at a single locus with pleiotropic effects. The independently occurring gene mutations of bm-2, bm-6, bm-22, and bm-33 are allelic. These chemically induced mutants had essentially identical EW structure, water loss, and cuticle deposition. Furthermore, 138 F2 plants from a bm-22 x WT-P954035 backcross showed no recombination of these traits. This unique mutation in a near-isogenic background provides a useful biological system to examine plant cuticle biosynthesis, physiology, and function.

Observations of Ice Nucleation and Propagation in Plants Using Infrared Video Thermography

We evaluated the use of infrared (IR) video thermography to observe directly ice nucleation and propagation in plants. An imaging radiometer with an HgCdTe long-wave (8–12 [mu]m) detector was utilized to image the thermal response of plants during freezing. IR images were analyzed in real time and recorded on videotape. Information on the videotape was subsequently accessed and analyzed utilizing IR image analysis software. Freezing of water droplets as small as 0.5 [mu]L was clearly detectable with the radiometer. Additionally, a comparison of temperature tracking data collected by the radiometer with data collected with thermocouples showed close correspondence. Monitoring of an array of plant species under different freezing conditions revealed that ice nucleation and propagation are readily observable by thermal imaging. In many instances, the ice nucleation-active bacterium Pseudomonas syringae placed on test plants could be seen to initiate freezing of the whole plant. Apparent ice nucleation by intrinsic nucleators, despite the presence of ice nucleation-active bacteria, was also evident in some species. Floral bud tissues of peach (Prunus persica) could be seen to supercool below the temperature of stem tissues, and ice nucleation at the site of insertion of the thermocouple was frequently observed. Rates of propagation of ice in different tissues were also easily measured by thermal imaging. This study demonstrates that IR thermography is an excellent method for studying ice nucleation and propagation in plants.

Cell shape and localisation of ice in leaves of overwintering wheat during frost stress in the field.

Wheat leaf pieces were excised and freeze-fixed in the field, preparatory to low-temperature scanning electron microscopy to study distribution of ice within leaf blades, and associated cell shapes, during natural frosts. Pieces of leaf blades from wheat plants (Triticum aestivum L. 7942H1-20-8) overwintering in Indiana, USA (January, 1991), were excised and immediately freeze-fixed by manually plunging in melting freon. Cells in controls were turgid and extracellular ice was absent. The leaves of the frost-stressed plants froze at about — 2.4° C, and at that temperature extracellular ice was mainly located sub-epidermally, including in the substomatal cavity, and occupied about 14% of the fracture faces. The frequency of ice particles per unit leaf area in two specimens was 14 and 210 · mm−2 (about 140 and 2100 · g−1 leaf fresh-weight basis). At -9.0° C, ice filled the extracellular spaces, occupying 61% of the fracture faces. Cells were somewhat collapsed at -2.4° C and were much more collapsed at -9.0° C. The epidermal cells were more collapsed than the mesophyll cells. Tissue structure (connections with adjacent cells), wall flexibility, and ice growth may all have influenced the shapes of the collapsing cells. The experiments demonstrate the feasibility of freeze-fixation in the field. The sub-epidermal location of most ice indicates that in the field either (i) ice is nucleated sub-epidermally (implying both the presence of nucleators and the presence of liquid water in the sub-epidermal spaces) or (ii) ice is nucleated on the leaf surface, then propagates into the leaf probably through stomata.

A comparison of seasonal ultrastructural changes in stem tissues of peach (Prunus persica) that exhibit contrasting mechanisms of cold hardiness

A comparative study of seasonal changes in cortical and xylem parenchyma cells of peach twigs (Prunus persica) indicated that, despite contrasting mechanisms of cold hardiness, both cell types exhibited analogous seasonal changes in cell fine structure coincident with seasonal stages of cold hardiness. Marked plasmalemma infoldings associated with multivesicular bodies (paramural bodies) and/or complex membrane aggregates (myelin-like bodies), and sometimes containing fibrillar wall material, were observed during periods of acclimation and deacclimation. Alteration of the plasmalemma by membrane turnover may occur during these periods.

Epicuticular Wax Morphology of Bloomless (bm) Mutants in Sorghum bicolor

Sorghum bicolor mutants for cuticular wax production provide a model system for analysis of epicuticular wax (EW) physiology, biochemistry, and genetics. Mutants produced from seeds treated with the chemical mutagens diethyl sulfate (DES) and ethyl methanesulfonate (EMS) were selected in the M2 generation and self-pollinated to produce near-isogenic mutants of two classes: bloomless (lacking visible EW) and sparse bloom (possessing little visible EW). Scanning electron microscopy was used to further divide 33 selected lines into 14 unique classes based on altered EW structure. Mutations have affected the structure of cork silica (CS) cell associated EW or both CS cell and cuticle EW. The resulting spectrum of altered EW structure indicates unique alterations in EW biosynthesis or deposition which may correlate with specific EW alleles and loci within the Sorghum genome.

Extracellular freezing in leaves of freezing-sensitive species

Abstract. Low-temperature scanning-electron microscopy was used to study the freezing of leaves of five species that have no resistance to freezing: bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.), cucumber (Cucumis sativus L.), and corn (Zea mays L.). In the leaves of the four dicotyledonous species, ice was extracellular and the cells of all tissues were collapsed. In contrast, in maize leaves ice was extracellular in the mesophyll, and these cells were collapsed, but the epidermal and bundle-sheath cells apparently retained their original shapes and volume. It is concluded that the leaves of the freezing-sensitive dicotyledonous species tested were killed by cellular dehydration induced by extracellular freezing, and not by intracellular freezing. Freezing injury in maize leaves apparently resulted from a combination of freezing-induced cellular dehydration of some cells and intracellular ice formation in epidermal and bundle-sheath cells.

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.

Mechanisms of Frost Survival and Freeze-Damage in Nature

Conifers experience a variety of frost scenarios and associated injuries in their natural range. For example, injury from spring frost is a frequent occurrence throughout the northern range of conifers — injuries are also caused by night frosts during the active growth period, when the tissues are sensitive to even slight freezing temperatures. However, night frosts during early autumn can also cause injuries if the weather conditions have not been favorable to cold acclimation. Although cold acclimated conifers from northern regions can usually tolerate severe freezing temperatures, winter desiccation and decreased cold hardiness due to unseasonable warm spells can cause injuries to conifer tissues during mid-winter and early spring. Thus, there are occasions when even the hardiest conifer species in the northern boreal forests experience injury of some sort.

Involvement of Cork Cells in the Secretion of Epicuticular Wax Filaments on Sorghum bicolor (L.) Moench

Tubular epicuticular wax (EW) filaments on Sorghum bicolor were shown to be secreted from smooth conical papillae within the apical walls of epidermal cork cells. Ultrastructural changes during light-induced EW secretion were examined in wild-type plants and near-isogenic mutants with reduced total EW deposition. Our results indicated that cork cell ER membranes were involved in the production of epicuticular wax precursors (EWPs). The density of ER increased during light exposure and preceded EW synthesis. The increase in ER was directly related to total EW deposition on wild-type and mutant abaxial sheaths. The orientation of ER membranes toward papillae secretion sites indicated that EWP may undergo ER-mediated directional transport. The high vesicle density in cytoplasmic extensions under papillae indicated that EWPs were vesiculated for exocytosis at the papillar secretion sites. Osmiophilic globules did not appear to be direct EWPs as previously reported. Osmiophilic globules in cork cells were never present in cell walls, cuticles, vesicles, or preferentially associated with ER; globules were randomly dispersed in the cytoplasm and rarely present during the EW-induction period. Distinct microchannels or pores were not evident in the cell wall or cuticle layers, indicating that EWPs diffused to the surface. Wall swellings near the base of papillae where a dense-staining wall modification first contacts the cuticle and where EW filaments emerge indicate a potential preferred pathway for EWP transport. An osmiophilic layer within apical cork cell walls appears to function in EW secretion; however, its exact role is yet unclear.

Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.