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- Author or Editor: Jeffrey A. Anderson x
- Journal of the American Society for Horticultural Science x
Acute high-temperature stress affects plant protein structure, leading to denaturation and aggregation. Although folding states of individual proteins have been extensively studied, little information is available on protein thermostability in complex mixtures. The objective of this study was to systematically examine protein stabilizing and destabilizing factors in pepper (Capsicum annuum L.) leaf extracts using light transmission measurements. Increasing turbidity and subsequent precipitation were monitored in heated extracts as changes in light scattering at 540 nm. Factors evaluated included leaf tissue concentration, buffer pH, compounds that can stabilize enzymatic activity (chelating agent, complexer of phenolics, and a compatible solute), and destabilizing agents (nonionic detergent and divalent cation). Leaf extract thermostability decreased with increasing tissue concentration from 6 to 60 g fresh weight per liter of buffer. Turbidity and precipitation occurred after exposure to higher temperatures as buffer pH increased from 6.0 to 7.0. Ethylenediaminetetraacetic acid (chelating agent) and polyvinylpolypyrrolidone (complexer of alkaloids and phenolics) had relatively small effects on extract thermostability. Nonionic detergent (Tween 20) destabilized extract thermostability, especially when incorporated in the extraction buffer. Calcium reduced thermostability by about 2 °C when added as CaCl2 at 1 mm. Calcium caused an increase in turbidity that was not directly associated with protein complexes and was not affected by treatment temperature. Mannitol, a compatible solute, increased the temperature at which turbidity and precipitation were induced, but only at high (500 mm) concentrations. Agents that stabilize or destabilize proteins at high temperatures can be assayed in plant extracts by measuring turbidity changes at 540 nm. These findings can be applied to functional studies determining the basis for differences in thermotolerance between genotypes and between control and acclimated tissues.
`Early Calwonder' pepper (Capsicum annuum L.) and `Jubilee' corn (Zea mays L.) leaf disks exposed to high temperature stress produced ethylene, ethane, methanol, acetaldehyde, and ethanol based on comparison of retention times during gas chromatography to authentic standards. Methanol, ethanol, and acetaldehyde were also identified by mass spectroscopy. Corn leaf disks produced lower levels of ethylene, ethane, and methanol, but more acetaldehyde and ethanol than pepper. Production of ethane, a by-product of lipid peroxidation, coincided with an increase in electrolyte leakage (EL) in pepper but not in corn. Compared with controls, pepper leaf disks infiltrated with linolenic acid evolved significantly greater amounts of ethane, acetaldehyde, and methanol and similar levels of ethanol. EL and volatile hydrocarbon production were not affected by fatty acid infiltration in corn. Infiltration of pepper leaves with buffers increasing in pH from 5.5 to 9.5 increased methanol production.
Although heat stress injury is known to be associated with membrane dysfunctions, protein structural changes, and reactions of activated forms of oxygen, the underlying mechanisms involved are poorly understood. In this study, the relationships between thermotolerance and hydrogen peroxide (H2O2) defense systems, radical scavenging capacity [based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) reduction], and protein aggregation were examined in vinca [Catharanthus roseus (L.) G. Don `Little Bright Eye'], a heat tolerant plant, and sweet pea (Lathyrus odoratus L. `Explorer Mix'), a heat susceptible plant. Vinca leaves were 5.5 °C more thermotolerant than sweet pea leaves based on electrolyte leakage analysis. Vinca leaf extracts were more resistant to protein aggregation at high temperatures than sweet pea leaf extracts, with precipitates forming at ≥40 °C in sweet pea and at ≥46 °C in vinca. Vinca leaves also had nearly three times greater DPPH radical scavenging capacity than sweet pea leaf extracts. Two enzymatic detoxifiers of H2O2, catalase (CAT) and ascorbate peroxidase (APOX), demonstrated greater activities in vinca leaves than in sweet pea leaves. In addition, CAT and APOX were more thermostable in vinca, compared with sweet pea leaves. However, tissue H2O2 levels did not differ between controls and tissues injured or killed by heat stress in either species, suggesting that H2O2 did not play a direct role in acute heat stress injury in vinca or sweet pea leaves. Greater thermotolerance in vinca, compared with sweet pea, was associated with greater DPPH radical scavenging capacity, indicating that AOS other than H2O2 may be involved in acute heat stress injury.
Electrolyte leakage (EL) and ethane: ethylene ratio (EER) responses of pepper (Capsicum annuum L. `Early Calwonder') leaf disks to temperature stresses were in close agreement. Midpoints of sigmoidal response curves following freezing stress were -4.6 and -4.4C for EL and EER, respectively, and 49.0 and 48.7C following high-temperature stress. Leaf disks exposed to temperatures below -4C in freezing experiments were induced to freeze while disks held at -4C and higher avoided freezing by supercooling. Evolution of ethane and EL were measured from disks infiltrated with a saturation series of 18-C fatty acids ranging from 0 to 3 double bonds. Only cis-9,12,15 linolenic acid (18:3 n-3) stimulated ethane production and EL. In a second fatty acid experiment with 18 and 20-C acids with a double bond 3 (n-3) or 6 (n-6) carbons from the nonpolar end of the molecule, n-3 fatty acids stimulated more ethane than n-6 acids with the same number of carbons. Trienoic 18-C fatty acids stimulated more ethane than trienoic 20-C acids. Both 18-C trienoic acids yielded significantly greater EL, while values from 20-C fatty acids were only slightly higher than those of controls. Propyl gallate, a free radical scavenger, reduced ethane production without decreasing EL or K+ leakage.
Cold acclimation (CA) of `Midiron' and `Tifgreen' turf bermudagrasses (Cynodon dactylon L. Pers. × C. transvaalensis Burtt-Davy) induced tolerance to lower freezing temperatures and altered protein synthesis in crowns. LT50 (lethal temperature for 50% of plants) values were lowered ≈5C after 4 weeks in controlled-environment chambers under CA [8/2C (day/night) cycles with a 10-hour photoperiod] vs. non-CA (28/24C) conditions. LT50 values for `Midiron' plants decreased from -6.5 to -11.3C after CA and from -3.6 to -8.5C for `Tifgreen'. Proteins synthesized by isolated crowns were radiolabeled in vivo for 16 hours with 35 S-methionine and 35 S-cysteine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography revealed increased synthesis of several cold-regulated (COR) proteins in CA crowns of both cultivars. Synthesis of intermediate molecular weight (MW) (32 to 37 kDa) and low-MW (20 to 26 kDa) COR proteins was greater in `Midiron' than `Tifgreen' crowns.
Abstract
‘Orlando’ tangelo (Citrus reticulata Blanco × Citrus paradisi Macf.) trees not irrigated in the fall, but protected by under-tree sprinkling during a frost, sustained the lowest percentage of leaf and fruit damage as determined 6 weeks after the frost. Trees irrigated both in the fall and during a frost, or those receiving no fall irrigation or under-tree sprinkling, were intermediate in fruit damage. Fall irrigation without sprinkling the night of a frost contributed to the most severe damage to leaves and fruit. Soil moisture content of irrigated blocks was significantly greater than for non-irrigated blocks during the fall, yet afternoon leaf xylem water potential and stem water content were comparable. Leaf freezing point of detached leaves of ‘Orlando’ and navel orange (Citrus sinensis (L.) Osbeck) was poorly correlated with leaf xylem water potential, abaxial diffusion resistance, and relative water content. Leaf freezing and killing temperature was unaffected by fall irrigation and ranged from -5.8 to -6.8°C from October until December in 1978 and 1979.
The hypersensitive response in resistant plants exposed to incompatible pathogens involves structural changes in the plant cell wall and plasma membrane. Cell wall changes may include pectin deesterification resulting in release of methanol. The time course of methanol production was characterized from `Early Calwonder 20R' pepper (Capsicum annuum L.) leaves infiltrated with the incompatible pathogen, Xanthomonas campestris pv. vesicatoria (Doidge) Dye race 1 (XCV). In the first time course experiment, leaves were infiltrated with either 108 colony-forming units/mL of XCV or water control. Leaf panels (1 × 5 cm) were excised after dissipation of water soaking, then incubated in vials at 24 °C. Headspace gas was analyzed at 6-hour intervals up to 24 hours. The rate of methanol production from resistant pepper leaves infiltrated with XCV was greatest during the first 12 hours after excision. In another experiment, leaf panels were harvested at 6-hour intervals up to 24 hours after inoculation and incubated for 12 hours at 24 °C to determine the relationship between the interval from inoculation to leaf excision and methanol production. The highest rate of methanol production was obtained when the interval between bacterial infiltration and leaf excision was 18 hours. The relationship between methanol release and changes in the degree of methylesterification (DOM) of cell wall pectin was determined in near isogenic lines of `Early Calwonder' pepper plants resistant (20R) and susceptible (10R) to XCV race 1. Cell walls were prepared from resistant and susceptible pepper leaves infiltrated with XCV or water. XCV-treated resistant leaves had 18% DOM and 9.7 nmol·g-1·h-1 of headspace methanol, and the susceptible leaves had 48% DOM with 0.2 nmol·g-1·h-1 methanol. Susceptible and resistant control leaves infiltrated with water had 55% and 54% DOM, respectively, with no detectable methanol production. Increased methanol production in resistant pepper leaves inoculated with XCV coincided with an increase in cell wall pH. Intercellular washing fluid of resistant pepper leaves had a significantly higher pH (6.9) compared to susceptible leaves (pH 5.1) and control leaves infiltrated with water (pH 5.1). Both 10R and 20R pepper leaves infiltrated with buffer at increasing pH's of 5.1, 6.9 or 8.7 had increased methanol production. Since deesterified pectin is more susceptible to degradation, demethylation may facilitate formation of pectic oligomers with defensive signalling activity.