Dale Haskell

Exploring the Temperature-Stress Metabolome of Arabidopsis

Metabolic profiling analyses were performed to determine metabolite temporal dynamics associated with the induction of acquired thermotolerance in response to heat shock and acquired freezing tolerance in response to cold shock. Low-Mr polar metabolite analyses were performed using gas chromatography-mass spectrometry. Eighty-one identified metabolites and 416 unidentified mass spectral tags, characterized by retention time indices and specific mass fragments, were monitored. Cold shock influenced metabolism far more profoundly than heat shock. The steady-state pool sizes of 143 and 311 metabolites or mass spectral tags were altered in response to heat and cold shock, respectively. Comparison of heat- and cold-shock response patterns revealed that the majority of heat-shock responses were shared with cold-shock responses, a previously unknown relationship. Coordinate increases in the pool sizes of amino acids derived from pyruvate and oxaloacetate, polyamine precursors, and compatible solutes were observed during both heat and cold shock. In addition, many of the metabolites that showed increases in response to both heat and cold shock in this study were previously unlinked with temperature stress. This investigation provides new insight into the mechanisms of plant adaptation to thermal stress at the metabolite level, reveals relationships between heat- and cold-shock responses, and highlights the roles of known signaling molecules and protectants.

Characterization of a spinach gene responsive to low temperature and water stress

The characterization of a cDNA for an 85 kDa spinach protein, CAP85 (cold acclimation protein) that is responsive to cold acclimation and water stress is described. Both transcript and protein levels are increased during cold acclimation and water stress. A novel characteristic of CAP85 is the presence of an 11 amino acid, lysine-rich repeat, common to Group 2 LEAs (late embryogenesis abundant proteins), which is included within a larger repeating motif present in 11 copies. Two other motifs of 8 and 16 residues are also found in three and four copies, respectively. CAP85 like other dehydrins and cold-regulated polypeptides remains soluble upon boiling. Protein blot analyses indicate that CAP85 protein is expressed in all aerial tissues as well as in roots. RNA blots show the presence of mRNA for the 85 kDa protein in leaf, petiole, and root tissue. Cell fractionation studies suggest that CAP85 is predominately found in the cytosol.

Hydration-state-responsive proteins link cold and drought stress in spinach

Spinach (Spinacia oleracea L.) seedlings exposed to low nonfreezing temperatures (0–10° C) that promote cold acclimation, synthesize a variety cold-acclimation proteins and at the same time acquire a greater ability to withstand cellular dehydration imposed by the freezing of tissue water. Two of these proteins (160 and 85 kDa) become more abundant over time at low temperature. In addition, a small decline in tissue water status from a maximally hydrated state also appears to be associated with an initiation of the accumulation of these proteins at a noninductive temperature. Imposing a severe water stress on young seedlings grown at 25° C by withholding water leads to substantial accumulation of the 160- and 85-kDa proteins, and maximal induction of freezing tolerance. This evidence implies that responses to cold acclimation and water stress involve common mechanisms, and further establishes the linkage of these two proteins with stresses having an osmotic component.

Coordinate and non-coordinate expression of the stress 70 family and other molecular chaperones at high and low temperature in spinach and tomato

Stress 70 molecular chaperones are found in all the major subcellular compartments of plant cells, and they are encoded by a multigene family. Twelve members of this family have been identified in spinach. The expression of the stress 70 molecular chaperones in response to heat shock is well-known and it appears that low temperature exposure can also stimulate their expression. However, it has been difficult to determine which member(s) of the family are specifically responsive to low temperature. This study was initiated to determine the levels of expression of the stress 70 family members and other selected chaperones in response to high and low temperature exposure. During heat shock of spinach, of the 10 stress 70 family members that were examined, all 10 showed increased RNA levels after one hour, and all showed down-regulation at longer durations of high temperature exposure. However, the response to low temperature was quite variable and complex. Some members were induced, some were transiently up-regulated, while others showed sustained up-regulation at a low non-freezing temperature. In comparison, the entirety of the molecular chaperone expression response of cold-sensitive tomato at the same low non-freezing temperature was even more dramatic with 11 of 15 molecular chaperones tested exhibiting elevated expression. The increased chaperone expression is consistent with the hypothesis that the biogenesis or stability of some proteins is compromised at low non-freezing temperatures. In contrast, mild freezing sufficient to cause injury of spinach did not materially activate chaperone expression.

Changes in freezing tolerance and polypeptide content of spinach and citrus at 5 °C☆☆☆

Abstract The freezing tolerance of spinach leaf, petiole, hypocotyl, and root tissues and citrus leaf tissue was determined before and after 1 week of cold acclimation at 5 °C. Spinach leaf, petiole, and hypocotyl tissues increased in freezing tolerance upon exposure to 5 °C, while root tissue failed to show significant increased freezing tolerance. Citrus leaf tissue also increased in freezing tolerance following exposure to 5 °C. The protein composition of nonacclimated and cold-acclimated spinach leaf, hypocotyl, and root tissues and citrus leaf tissue was studied using two-dimensional gel electrophoresis and silver staining. Spinach leaf tissue exposed to 5 °C contained two cold-acclimation polypeptides (CAPs) (Mr 160,000 and 85,000) not found in nonacclimated leaf tissue. Numerous other changes in the polypeptide composition of spinach leaf tissue were observed following cold acclimation, but the appearance of CAPs 160 and 85 were the most consistent in all experiments. Cold-acclimated citrus leaf tissue also contained a polypeptide (Mr 160,000) not found in nonacclimated citrus leaf tissue. The similarity in molecular weight and isoelectric point between the CAP 160 of citrus and spinach suggests that these widely unrelated species may synthesize the same protein in response to exposure to low temperature. In contrast, both nonacclimated and cold-acclimated spinach hypocotyl tissue contained CAPs 85 and 160. However, cold-acclimated hypocotyls appeared to have concentrations of CAPs 85 and 160 higher than those of nonaccclimated hypocotyls. Protein analysis of nonacclimated and cold-acclimated spinach root tissue revealed no major qualitative differences. Furthermore, roots exposed to 5 °C did not synthesize proteins analogous to any of the CAPs synthesized in leaf tissue at low temperature. The inability of root tissue to synthesize CAPs at low temperature may be a factor in the lack of root freezing tolerance.

Differential Influence of ATP on Native Spinach 70-Kilodalton Heat-Shock Cognates

A constitutively expressed class of 70-kD heat-shock cognate (HSC70) proteins from spinach leaf tissue was purified based on their affinity for ATP-agarose. The affinity-purified spinach proteins were resolved into at least three different forms on two-dimensional gels. Under native conditions, and in the absence of ATP, the affinity-purified proteins were separated into three molecular mass classes by gel-filtration chromatography; a monomer of 85 kD, a multimer of 280 kD, and a large molecular mass oligomer of >650 kD. All molecular mass forms contained a major protein that migrated at 79 kD on sodium dodecyl sulfate-polyacrylamide gels. N-terminal sequencing of the 79-kD purified monomer showed the highest homology to the endoplasmic reticulum-luminal HSC70. Addition of Mg-ATP to monomeric HSC70 did not alter its migration during gel filtration. Addition of Mg-ATP to the dimer converted it to monomer and oligomeric forms, whereas the presence of ATP converted a fraction of the large molecular mass oligomeric form of HSC70 to dimeric and monomeric forms. Only the large molecular mass oligomeric HSC70 appears to autophosphorylate in vitro in the presence of [[gamma]-32P]- ATP. Dimers and monomers can bind ATP by a nonhydrolytic mechanism and undergo a conformational change in the presence of Mg-ATP. In this paper we discuss the effects that ATP may have on the regulation of plant HSC70.

Caps, Cors, Dehydrins, and Molecular Chaperones: Their Relationship with Low Temperature Responses in Spinach

When exposed to low non-freezing temperatures (0–10°C), the freezing tolerance of spinach increases in a time dependent fashion from an LT50 of -4°C to -14°C. Associated with the low temperature induction of freezing tolerance is the increased expression of at least 20 proteins. Three of these low temperature responsive proteins were selected for study and found to belong to two families of stress proteins, the LEA/dehydrin/Rab and the heat shock 70s. Their possible role in low temperature tolerance mechanisms and a model for the role of ATP in peptide binding activities of the 70 kDa heat shock molecular chaperones are discussed.

Molecular Chaperones: Do they Have a Role in Cold Stress Responses of Plants?

Molecular chaperones are proteins that assist in the in vivo biogenesis of enzymes and structural proteins. They participate in biogenesis in several ways by: binding to non-native nascent peptides emerging from ribosomes thereby preventing irreversible aggregation prior to folding, maintaining translocation across organelle membranes by stabilizing unfolded translocation competent forms, and helping in the assembly of oligomeric complexes. Numerous aspects of these processes are sensitive to high temperatures and consequently many molecular chaperones were first characterized as heat shock proteins. Generally lower temperatures increase the stability of proteins favoring the native state. However, there is a theoretical basis for a decreased stability and denaturation of some, so called “cold labile” proteins, and some aspects of translocation and assembly may also be similarly influenced by low temperature. The dehydration stress imposed during a freeze/ thaw cycle may further alter the intracellular milieu in ways that could favor protein denaturation. An examination of the RNA levels of several members of one family of molecular chaperones, the HSP70s, in response to exposure of spinach to 5°C revealed a pattern of differential expression that is consistent with a hypothesis that suggests that certain components of the protein biogenesis machinery requires some level of augmentation. It is proposed that chilling injury may arise, in part, from an impairment of normal protein biogenesis leading to an inability to form, or maintain, functional enzymes and structural proteins essential for cell homeostasis. Since the native state is stabilized at low temperature for most proteins, abnormalities in protein biogenesis would not be a global consequence, but only affect a subset of the proteins present in chilling sensitive plants.