SUMMARYIn nature, plants have to cope with a wide range of stress conditions that often occur simultaneously or in sequence. To investigate how plants cope with multi-stress conditions, we analyzed the dynamics of whole-transcriptome profiles of Arabidopsis thaliana exposed to six sequential double stresses inflicted by combinations of: (i) infection by the necrotrophic fungus Botrytis cinerea, (ii) herbivory by chewing larvae of Pieris rapae, and (iii) drought stress. Each of these stresses induced specific expression profiles over time, in which one-third of all differentially expressed genes was shared by at least two single stresses. Of these, 394 genes were differentially expressed during all three stress conditions, albeit often in opposite directions. When two stresses were applied in sequence, plants displayed transcriptome profiles that were very similar to the second stress, irrespective of the nature of the first stress. Nevertheless, significant first-stress signatures could be identified in the sequential stress profiles. Bioinformatic analysis of the dynamics of coexpressed gene clusters highlighted specific clusters and biological processes of which the timing of activation or repression was altered by a prior stress. The first-stress signatures in second stress transcriptional profiles were remarkably often related to responses to phytohormones, strengthening the notion that hormones are global modulators of interactions between different types of stress. Because prior stresses can affect the level of tolerance against a subsequent stress (e.g. prior herbivory strongly affected resistance to B. cinerea), the first-stress signatures can provide important leads for the identification of molecular players that are decisive in the interactions between stress response pathways.

The complete mitochondrial (mt) genome of Crossostoma lacustre, a freshwater loach from mountain stream of Taiwan, has been cloned and sequenced. This fish mt genome, consisting of 16558 base-pairs, encodes genes for 13 proteins, two rRNAs, and 22 tRNAs, in addition to a regulatory sequence for replication and transcription (D-loop), is similar to those of the other vertebrates in both the order and orientation of these genes. The protein-coding and ribosomal RNA genes are highly homologous both in size and composition, to their counterparts in mammals, birds, amphibians, and invertebrates, and using essentially the same set of codons, including both the initiation and termination signals, and the tRNAs. Differences do exist, however, in the lengths and sequences of the D-loop regions, and in space between genes, which account for the variations in total lengths of the genomes. Our observations provide evidence for the first time for the conservation of genetic information in the fish mitochondrial genome, especially among the vertebrates.

SummaryIn nature, plants are exposed to biotic and abiotic stresses that often occur simultaneously. Therefore, plant responses to combinations of stresses are most representative of how plants respond to stresses.We used RNAseq to assess temporal changes in the transcriptome of Arabidopsis thaliana to herbivory by Pieris rapae caterpillars, either alone or in combination with prior exposure to drought or infection with the necrotrophic fungus Botrytis cinerea.Pre-exposure to drought stress or Botrytis infection resulted in a significantly different timing of the caterpillar-induced transcriptional changes. Additionally, the combination of drought and P. rapae induced an extensive downregulation of A. thaliana genes involved in defence against pathogens. Despite a more substantial growth reduction observed for plants exposed to drought plus P. rapae feeding compared with P. rapae feeding alone, this did not affect weight increase of this specialist caterpillar.Plants respond to combined stresses with phenotypic and transcriptional changes that differ from the single stress situation. The effect of a previous exposure to drought or B. cinerea infection on transcriptional changes to caterpillars is largely overridden by the stress imposed by caterpillars, indicating that plants shift their response to the most recent stress applied.

The distribution of a heavy metal binding protein, metallothionein, was studied immunocytochemically by using antimetallothionein antibody and the immunoperoxidase staining technique on histological sections of liver, kidney, intestine, lung, and testis from cadmium-treated rats. These tissues either accumulate heavy metals (e.g., liver, kidney, and testis) or are exposed to metal by ingestion or inhalation (intestine and lung). Staining for metallothionein was Because of the secretory, absorptive, or nutritive function of the metallothionein-localizing cells in the organs studied, we suggest that metallothionein may be involved in metal storage or transport in addition to its commonly proposed detoxification role.Metallothionein-a low molecular weight (Mr 6,100), sulfurrich protein-has been identified in a wide variety oforganisms ranging from yeast to man (1, 2). The molecule is characterized by a high cysteine content (20 of 61 amino acids in mammalian forms) and the ability to bind class II B transition metal atoms (e.g., Zn, Cd, or Hg). The precise biological function of metallothionein is not known, although it has been suggested to be important in metal homeostasis and detoxification because of its presence in many mammalian tissues (1-3). In addition, metallothionein synthesis has been shown to increase in the kidney (4), liver (4, 5), lung (6), and intestine (7) ofrodents after treatment with heavy metals. Stressful environmental conditions such as food restriction (8, 9), cold exposure (10), or physical exercise (10) have also been reported to increase metallothionein content in liver tissue. Furthermore, treatment ofcells in culture or experimental animals with glucocorticoid hormones can also induce metallothionein synthesis (9,11,12 MATERIALS AND METHODS Experimental Animals. Inbred male McCollum rats (6-8 mo of age; 450-500 g of body weight) were kept in animal quarters on a light/dark cycle (12 hr/12 hr) and were fed Purina chow and water ad lib. For induction ofmetallothionein synthesis the animals were injected subcutaneously with cadmium chloride in saline (1 mg/kg of body weight) once daily during the early afternoon for 4 consecutive days. The animals were sacrificed 48 hr after the last injection.Metallothionein. Rat metallothionein was isolated from liver and purified as described (13). Isometallothioneins A and B were mixed in equimolar proportions and crosslinked with glutaraldehyde prior to injection into rabbits for antibody production. The preparation and characterization of the metallothionein antibody have been described (13).Tissue Preparation. Experimental animals were sacrificed by cervical dislocation. Organs of interest were removed, washed briefly in isotonic saline, and fixed in Helly's fluid for 6-8 hr, except testis, which was fixed in Bouin's fixative for 24 hr. The tissue fragments were dehydrated by a series ofchanges in dioxane, transferred to toluene/terpineol (3:1), and embedded in paraffin. Sections were cut at 6-8 tum and mounted on slides previously coate...