Charles L. Guy

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.

Acquired tolerance to temperature extremes

Acquired tolerance to temperature stresses is a major protective mechanism. Recent advances have revealed key components of stress signal transduction pathways that trigger enhanced tolerance, and several determinants of acquired tolerance have been identified. Although high and low temperature stresses impose different metabolic and physical challenges, acquired tolerance appears to involve general as well as stress-specific components. Transcriptome studies and other genomic-scale approaches have accelerated the pace of gene discovery, and will be invaluable in efforts to integrate all the different protective and repair mechanisms that function in concert to confer acquired tolerance.

β-Amylase Induction and the Protective Role of Maltose during Temperature Shock

A number of studies have demonstrated β-amylase induction in response to abiotic stress. In the present work, a temperature response profile in 5°C increments from 45°C to 0°C showed that induction at temperature extremes was specific for two members of the gene family (BMY7 and BMY8). Both members encode proteins that possess apparent transit peptides for chloroplast stromal localization. However, induction was not observed for other key starch degrading enzymes demonstrating a rather specific response to temperature stress for BMY7 and BMY8. Time course experiments for heat shock at 40°C and cold shock at 5°C showed that β-amylase induction correlated with maltose accumulation. Maltose has the ability, as demonstrated by in vitro assays, to protect proteins, membranes, and the photosynthetic electron transport chain at physiologically relevant concentrations. Therefore, β-amylase induction and the resultant maltose accumulation may function as a compatible-solute stabilizing factor in the chloroplast stroma in response to acute temperature stress.

Phylogeny of the American Amaryllidaceae based on nrDNA ITS sequences

Abstract Analysis of three plastid DNA sequences for a broad sampling of Amaryllidaceae resolve the American genera of the Amaryllidaceae as a clade that is sister to the Eurasian genera of the family, but base substitution rates for these genes are too low to resolve much of the intergeneric relationships within the American clade. We obtained ITS rDNA sequences for 76 species of American Amaryllidaceae and analyzed the aligned matrix cladistically, both with and without gaps included, using two species of Pancratium as outgroup taxa. ITS resolves two moderately to strongly supported groups, an Andean tetraploid clade, and a primarily extra-Andean “hippeastroid” clade. Within the hippeastroid clade, the tribe Griffineae is resolved as sister to the rest of Hippeastreae. The genera Rhodophiala and Zephyranthes are resolved as polyphyletic, but the possibility of reticulation within this clade argues against any re-arrangement of these genera without further investigation. Within the Andean subclade, Eustephieae resolves as sister to all other tribes; a distinct petiolate-leafed group is resolved, combining the tribe Eucharideae and the petiolate Stenomesseae; and a distinct Hymenocallideae is supported. These Andean clades are all at least partially supported by plastid sequence data as well. We infer from our data that a great deal of the diversity of the family in the Americas is recent, and that the American Amaryllidaceae may have been reduced to peripheral isolates some time after its initial entry and spread through the Americas. While the sister relationship of the American and Eurasian clades might argue for a Boreotropical origin for the family in America, the cladistic relationships within the American clade based on ITS do not provide any further support for this or any other hypothesis of the familys entry into America. The new tribe Clinantheae is described (four genera: Clinanthus, Pamianthe, Paramongaia, and Pucara), and the lorate-leafed species of Stenomesson are transferred to Clinanthus. Communicating Editor: Kathleen A. Kron

Physiological and Molecular Assessment of Altered Expression of Hsc70-1 in Arabidopsis. Evidence for Pleiotropic Consequences

Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44°C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.

Glucan, Water Dikinase Activity Stimulates Breakdown of Starch Granules by Plastidial β-Amylases

Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them β-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized β-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.

The Organization and Evolution of the Spinach Stress 70 Molecular Chaperone Gene Family

The stress 70 molecular chaperones of plants are localized and function in all of the major subcellular compartments of the cell. Collectively, all of the various forms are encoded by a multigene family in the nucleus. At least 12 members of this family have been found, and sequence and DNA blot analyses provide an emerging description of the diversity of gene structure organization for this family of evolutionarily conserved proteins in spinach. They exhibit not only structural diversity in the organization of coding and noncoding regions but also distinct expression patterns for different tissues and abiotic conditions. The results of phylogenetic analyses are concordant with at least four major evolutionary events that gave rise to stress 70 molecular chaperones in each of four major subcellular compartments of plant cells: the plastid, mitochondrion, cytoplasm, and endoplasmic reticulum. The varied expression patterns also illustrate the complexity of effectively interpreting the role of any one of these stress-related proteins in response to abiotic stress in the absence of context to the other members of the family.

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC1 progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.

Non-linear PCA: a missing data approach

MOTIVATION Visualizing and analysing the potential non-linear structure of a dataset is becoming an important task in molecular biology. This is even more challenging when the data have missing values. RESULTS Here, we propose an inverse model that performs non-linear principal component analysis (NLPCA) from incomplete datasets. Missing values are ignored while optimizing the model, but can be estimated afterwards. Results are shown for both artificial and experimental datasets. In contrast to linear methods, non-linear methods were able to give better missing value estimations for non-linear structured data. APPLICATION We applied this technique to a time course of metabolite data from a cold stress experiment on the model plant Arabidopsis thaliana, and could approximate the mapping function from any time point to the metabolite responses. Thus, the inverse NLPCA provides greatly improved information for better understanding the complex response to cold stress. CONTACT scholz@mpimp-golm.mpg.de.

Systematics of Amaryllidaceae based on cladistic analysis of plastid sequence data

Cladistic analyses of plastid DNA sequences rbcL and trnL-F are presented separately and combined for 48 genera of Amaryllidaceae and 29 genera of related asparagalean families. The combined analysis is the most highly resolved of the three and provides good support for the monophyly of Amaryllidaceae and indicates Agapanthaceae as its sister family. Alliaceae are in turn sister to the Amaryllidaceae/Agapanthaceae clade. The origins of the family appear to be western Gondwanaland (Africa), and infrafamilial relationships are resolved along biogeographic lines. Tribe Amaryllideae, primarily South African, is sister to the rest of Amaryllidaceae; this tribe is supported by numerous morphological synapomorphies as well. The remaining two African tribes of the family, Haemantheae and Cyrtantheae, are well supported, but their position relative to the Australasian Calostemmateae and a large clade comprising the Eurasian and American genera, is not yet clear. The Eurasian and American elements of the family are each monophyletic sister clades. Internal resolution of the Eurasian clade only partially supports currently accepted tribal concepts, and few conclusions can be drawn on the relationships of the genera based on these data. A monophyletic Lycorideae (Central and East Asian) is weakly supported. Galanthus and Leucojum (Galantheae pro parte) are supported as sister genera by the bootstrap. The American clade shows a higher degree of internal resolution. Hippeastreae (minus Griffinia and Worsleya) are well supported, and Zephyranthinae are resolved as a distinct subtribe. An Andean clade marked by a chromosome number of 2n = 46 (and derivatives thereof) is resolved with weak support. The plastid DNA phylogenies are discussed in the context of biogeography and character evolution in the family.