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.
Jeffrey A. Anderson
Jeffrey A. Anderson
Chemical chaperones (CC) are plant stress-related compounds that can stabilize protein structure in adverse environments. Modes of action are thought to involve hydrogen bonding, primarily with the solvent, and hydrophobic stabilization of the protein core. The objective of this study was to determine structure–function relationships between CC (and structurally related compounds) and thermal stability of pepper (Capsicum annuum L.) leaf proteins. Both polarity [based on log Kow (the oil–water partition coefficient)] and capacity for hydrogen bonding (based on the number of OH groups) contributed to whether low-molecular-weight alcohols and polyols stabilized or destabilized proteins at elevated temperatures. Thermal stability increased with increasing number of OH groups at a fixed number of carbon atoms per molecule. Conversely, thermal stability decreased with increasing number of carbon atoms with a fixed number of OH groups. When CC solution concentrations were adjusted to the same concentration of OH groups (1.51 × 1022 OH groups per milliliter), protein thermal stability increased with increasing CC polarity. Mixtures of different CC had additive effects on increasing protein thermostability, but mixtures of stabilizing (mannitol) and destabilizing (methanol) compounds negated each other. As a strategy for increasing plant thermotolerance, identification and removal of destabilizing compounds should be equally effective as increasing levels of stabilizers in protecting protein conformation at elevated temperatures.
Jeffrey A. Anderson
`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.
Jeffrey A. Anderson and Julia Whitworth
Frostgard did not effectively promote the supercooling of flowering `Arking' strawberry (Fragaria ×ananassa Duch.) plants in the presence or absence of ice-nucleation-active bacteria when applied as a spray in laboratory experiments. Frostgard effectively promoted supercooling and reduced the ice propagation rate of aqueous solutions. Detached leaves infiltrated with Frostgard exhibited a negative linear relationship between freezing temperature and Frostgard concentrations from 0% to 20% (by volume). Leaves infiltrated with 20% Frostgard supercooled 1.7C lower than those infiltrated with distilled water. Ice propagation barriers in strawberry plants were observed. Individual leaves froze independently, and a thermal ice propagation barrier sometimes was observed at the crown.
Geeta K. Nanaiah and Jeffrey A. Anderson
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, and 49.0 and 48.8C following high temperature stress. Evolution of ethane and EL were measured from disks infiltrated with a saturation series of 18-carbon fatty acids ranging from 0 to 3 double bonds. Only linolenic acid (18:3 n-3) stimulated ethane production and EL. In a second fatty acid experiment with 18- and 20-carbon 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-carbon fatty acids stimulated more ethane than trienoic 20-carbon acids. Both 18-carbon acids yielded significantly greater EL than controls. Propyl gallate, a free radical scavenger, reduced ethane production without decreasing EL or K+ leakage.
Jeffrey A. Anderson and Sonali R. Padhye
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.
Geeta K. Nanaiah and Jeffrey A. Anderson
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.
B. Warren Roberts and Jeffrey A. Anderson
Experiments were conducted from 1989 to 1991 to compare the effectiveness of various cultural techniques in reducing solar injury (SI) and increasing yield of bell pepper (Capsicum annuum var. annuum `California Wonder') in southern Oklahoma. Treatments included black plastic mulch, white plastic mulch, straw mulch, living rye, spunbonded polypropylene used as a plant canopy shade, and bare soil. Marketable yields from plots shaded with spunbonded polypropylene rowcovers were equal to or greater than those from other treatments each year. Two out of 3 years, plots with a black plastic soil mulch had marketable yields lower than those from other treatments. SI was reduced by rowcover shade.